Template:Kyoto/Notebook/DMS
From 2014.igem.org
(123 intermediate revisions not shown) | |||
Line 127: | Line 127: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td> | + | <td>pSB1C3</td> |
<td>13.3</td> | <td>13.3</td> | ||
<td>1.90</td> | <td>1.90</td> | ||
Line 151: | Line 151: | ||
<th>Total/(µL)</tr> | <th>Total/(µL)</tr> | ||
<tr> | <tr> | ||
- | <td>8/1 | + | <td>8/1 pSB1C3 gel extraction product 2047bp</td> |
<td>1.5</td> | <td>1.5</td> | ||
<td>8/1 dddD gel extraction product 2545bp</td> | <td>8/1 dddD gel extraction product 2545bp</td> | ||
Line 174: | Line 174: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td> | + | <td>pSB1C3-dddD</td> |
<td>3</td> | <td>3</td> | ||
<td>Self-Making Competent Cells (DH5α)</td> | <td>Self-Making Competent Cells (DH5α)</td> | ||
Line 415: | Line 415: | ||
</tr> | </tr> | ||
</table> | </table> | ||
- | |||
<img src="https://static.igem.org/mediawiki/2014/4/46/Kyoto-dms-0803_electrophoresis_No2.jpg"> | <img src="https://static.igem.org/mediawiki/2014/4/46/Kyoto-dms-0803_electrophoresis_No2.jpg"> | ||
- | |||
<h4>Gel Extraction</h4> | <h4>Gel Extraction</h4> | ||
<span class="kyoto-author">Tatsui</span> | <span class="kyoto-author">Tatsui</span> | ||
Line 463: | Line 461: | ||
</table> | </table> | ||
<img src="https://static.igem.org/mediawiki/2014/b/bc/Kyoto-dms-0803_Gel_Extraction_No1_after.jpg"> | <img src="https://static.igem.org/mediawiki/2014/b/bc/Kyoto-dms-0803_Gel_Extraction_No1_after.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/d/d4/Kyoto-dms-0803_electrophoresis_No1.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/7/7d/Kyoto-dms-0803_electrophoresis_No3.jpg"> | ||
<table> | <table> | ||
<tr> | <tr> | ||
Line 481: | Line 481: | ||
<td>1.54</td> | <td>1.54</td> | ||
<td>0.85</td> | <td>0.85</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Ligation</h4> | ||
+ | <span class="kyoto-author">no name</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Vector/(µL)</th> | ||
+ | <th colspan=2>Insert/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th colspan=2>Ligase/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</tr> | ||
+ | <tr> | ||
+ | <td>pSB1C3</td> | ||
+ | <td>7</td> | ||
+ | <td>dddD</td> | ||
+ | <td>7</td> | ||
+ | <td>2x Buffer</td> | ||
+ | <td>15</td> | ||
+ | <td>T4 Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>30</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pSB1C3</td> | ||
+ | <td>7</td> | ||
+ | <td>dddD</td> | ||
+ | <td>14</td> | ||
+ | <td>2x Buffer</td> | ||
+ | <td>22</td> | ||
+ | <td>T4 Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>44</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pSB1C3</td> | ||
+ | <td>7</td> | ||
+ | <td>dddD</td> | ||
+ | <td>-</td> | ||
+ | <td>2x Buffer</td> | ||
+ | <td>15</td> | ||
+ | <td>T4 Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>7</td> | ||
+ | <td>30</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Transformation</h4> | ||
+ | <span class="kyoto-author">no name</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>CompetentCells</th> | ||
+ | <th>Medium</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>(1)</td> | ||
+ | <td>Self-Making Competent Cells (DH5α)</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>(2)</td> | ||
+ | <td>Self-Making Competent Cells (DH5α)</td> | ||
+ | <td>CP</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
Line 533: | Line 600: | ||
<table> | <table> | ||
<tr> | <tr> | ||
- | <th> | + | <th>Lane</th> |
- | <th>dddD+pSB1C3(1)-1</ | + | <th>Sample</th> |
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>dddD+pSB1C3(1)-1</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 1,098: | Line 1,169: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | < | + | <td>8/18 pSB1C3</td> |
<td>105</td> | <td>105</td> | ||
<td>1.30</td> | <td>1.30</td> | ||
Line 1,104: | Line 1,175: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td>dddD</td> | + | <td>8/18 dddD</td> |
<td>105</td> | <td>105</td> | ||
<td>1.27</td> | <td>1.27</td> | ||
Line 1,228: | Line 1,299: | ||
<th>Total/(µL)</tr> | <th>Total/(µL)</tr> | ||
<tr> | <tr> | ||
- | <td>pSB1C3</td> | + | <td>pSB1C3 control</td> |
<td>10.8</td> | <td>10.8</td> | ||
<td>-</td> | <td>-</td> | ||
Line 1,447: | Line 1,518: | ||
</table> | </table> | ||
<img src="https://static.igem.org/mediawiki/2014/6/6f/Kyoto-dms-0825_electrophoresis_No1.jpg"> | <img src="https://static.igem.org/mediawiki/2014/6/6f/Kyoto-dms-0825_electrophoresis_No1.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/2/2a/Kyoto-dms-0825_Gel_Extraction_No1_after.jpg"> | ||
</div> | </div> | ||
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</tr> | </tr> | ||
</table> | </table> | ||
- | |||
<img src="https://static.igem.org/mediawiki/2014/5/5f/Kyoto-dms-0825_Gel_Extraction_No2_after.jpg"> | <img src="https://static.igem.org/mediawiki/2014/5/5f/Kyoto-dms-0825_Gel_Extraction_No2_after.jpg"> | ||
<img src="https://static.igem.org/mediawiki/2014/2/25/Kyoto-dms-0825_Gel_Extraction_No2_before.jpg"> | <img src="https://static.igem.org/mediawiki/2014/2/25/Kyoto-dms-0825_Gel_Extraction_No2_before.jpg"> | ||
Line 1,495: | Line 1,566: | ||
<td>1.3</td> | <td>1.3</td> | ||
<td>8/18 dddD gel extraction product</td> | <td>8/18 dddD gel extraction product</td> | ||
- | <td>0. | + | <td>0.71</td> |
<td>2x Buffer</td> | <td>2x Buffer</td> | ||
<td>5</td> | <td>5</td> | ||
Line 1,609: | Line 1,680: | ||
<td>30sec</td> | <td>30sec</td> | ||
<td>30sec</td> | <td>30sec</td> | ||
- | <td> | + | <td>140sec</td> |
</tr> | </tr> | ||
</table> | </table> | ||
Line 1,716: | Line 1,787: | ||
</tr> | </tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/a/a8/Kyoto-dms-0827_electrophoresis_No1.jpg"> | ||
<table> | <table> | ||
<tr> | <tr> | ||
Line 1,778: | Line 1,850: | ||
</tr> | </tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/1/1e/Kyoto-dms-0827_electrophoresis_No2.jpg"> | ||
</div> | </div> | ||
<div> | <div> | ||
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<table> | <table> | ||
<tr> | <tr> | ||
- | <th>2x | + | <th>2x KAPA HiFi HotStart ReadyMix /(µL)</th> |
<th>V Pre-dddD 62/(µL)</th> | <th>V Pre-dddD 62/(µL)</th> | ||
<th>Rv Spe1-dddD 68/(µL)</th> | <th>Rv Spe1-dddD 68/(µL)</th> | ||
Line 1,913: | Line 1,986: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td>pSB1C3 E,S gel extraction product</td> | + | <td>pSB1C3 E,S gel extraction product control</td> |
<td>4.7</td> | <td>4.7</td> | ||
<td>-</td> | <td>-</td> | ||
Line 1,938: | Line 2,011: | ||
<td>dddD 8/29 ligation product</td> | <td>dddD 8/29 ligation product</td> | ||
<td>10</td> | <td>10</td> | ||
- | <td> | + | <td>DH5α</td> |
<td>40</td> | <td>40</td> | ||
<td>50</td> | <td>50</td> | ||
Line 1,946: | Line 2,019: | ||
<td>8/29 ligation product control</td> | <td>8/29 ligation product control</td> | ||
<td>10</td> | <td>10</td> | ||
- | <td> | + | <td>DH5α</td> |
<td>40</td> | <td>40</td> | ||
<td>50</td> | <td>50</td> | ||
Line 1,954: | Line 2,027: | ||
<td>pSB1C3 control</td> | <td>pSB1C3 control</td> | ||
<td>1</td> | <td>1</td> | ||
- | <td> | + | <td>DH5α</td> |
<td>20</td> | <td>20</td> | ||
<td>21</td> | <td>21</td> | ||
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<table> | <table> | ||
<tr> | <tr> | ||
- | <th>Quick | + | <th>Quick Taq/(µL)</th> |
<th>VF2/(µL)</th> | <th>VF2/(µL)</th> | ||
<th>VR/(µL)</th> | <th>VR/(µL)</th> | ||
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<h4>Electrophoresis</h4> | <h4>Electrophoresis</h4> | ||
- | <span class= | + | <span class="kyoto-author">Matsumoto</span> |
<table> | <table> | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
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<tr><td>8</td><td>1kb ladder</td> | <tr><td>8</td><td>1kb ladder</td> | ||
</table> | </table> | ||
- | + | <img src="https://static.igem.org/mediawiki/2014/6/63/Kyoto-dms-0901_electrophoresis_No1.jpg"> | |
<h4>Colony PCR</h4> | <h4>Colony PCR</h4> | ||
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<table> | <table> | ||
<tr> | <tr> | ||
- | <th>Quick | + | <th>Quick Taq/(µL)</th> |
<th>VF2/(µL)</th> | <th>VF2/(µL)</th> | ||
<th>VR/(µL)</th> | <th>VR/(µL)</th> | ||
Line 2,137: | Line 2,210: | ||
<h4>Electrophoresis</h4> | <h4>Electrophoresis</h4> | ||
- | <span class= | + | <span class="kyoto-author">Matsumoto</span> |
<table> | <table> | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
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<tr><td>8</td><td>1kb ladder</td> | <tr><td>8</td><td>1kb ladder</td> | ||
</table> | </table> | ||
- | + | <img src="https://static.igem.org/mediawiki/2014/a/a1/Kyoto-dms-0901_electrophoresis_No2.jpg"> | |
</div> | </div> | ||
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<h4>Electrophoresis</h4> | <h4>Electrophoresis</h4> | ||
- | <span class= | + | <span class="kyoto-author">Mastumoto</span> |
<table> | <table> | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
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<tr><td>3</td><td>control</td> | <tr><td>3</td><td>control</td> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/a/ac/Kyoto-dms-0901_electrophoresis_No3.jpg"> | ||
- | + | <h4>Cloning</h4> | |
- | <h4>Cloning<h4> | + | <span class="kyoto-author">Yasuda</span> |
- | <span class= | + | |
<table> | <table> | ||
<tr> | <tr> | ||
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<th>Annealing</th> | <th>Annealing</th> | ||
<th>Extension</th> | <th>Extension</th> | ||
- | <th>Final | + | <th>Final Extension</th> |
<th>Cooling</th> | <th>Cooling</th> | ||
</tr> | </tr> | ||
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<td>2min10sec</td> | <td>2min10sec</td> | ||
<td>5min</td> | <td>5min</td> | ||
- | <td> | + | <td>Infinity</td> |
</tr> | </tr> | ||
</table> | </table> | ||
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<h4>Restriction Enzyme Digestion</h4> | <h4>Restriction Enzyme Digestion</h4> | ||
- | <span class= | + | <span class="kyoto-author">Matsumoto</span> |
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | ||
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</table> | </table> | ||
- | <h4>Cloning<h4> | + | <h4>Cloning</h4> |
- | <span class= | + | <span class="kyoto-author">Yasuda</span> |
<p>Master Mix(x4)</p> | <p>Master Mix(x4)</p> | ||
<table> | <table> | ||
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<th>Annealing</th> | <th>Annealing</th> | ||
<th>Extension</th> | <th>Extension</th> | ||
- | <th>Final | + | <th>Final Extension</th> |
<th>Cooling</th> | <th>Cooling</th> | ||
</tr> | </tr> | ||
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<td>2min10sec</td> | <td>2min10sec</td> | ||
<td>5min</td> | <td>5min</td> | ||
- | <td> | + | <td>Infinity</td> |
</tr> | </tr> | ||
</table> | </table> | ||
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<h4>Gel Extraction</h4> | <h4>Gel Extraction</h4> | ||
- | <span class= | + | <span class="kyoto-author">Yamauchi</span> |
<table> | <table> | ||
<tr><th>Lane</th><th>Sample</th></tr> | <tr><th>Lane</th><th>Sample</th></tr> | ||
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<tr><td>6</td><td>1kb ladder</td></tr> | <tr><td>6</td><td>1kb ladder</td></tr> | ||
</table> | </table> | ||
- | + | <img src="https://static.igem.org/mediawiki/2014/5/54/Kyoto-dms-0903_Gel_Extraction_No1_before.jpg"> | |
+ | <img src="https://static.igem.org/mediawiki/2014/8/8b/Kyoto-dms-0903_Gel_Extraction_No1_after.jpg"> | ||
<h4>DNA Purification</h4> | <h4>DNA Purification</h4> | ||
- | <span class= | + | <span class="kyoto-author">Matsumoto</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
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<h4>Ligation</h4> | <h4>Ligation</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda, Li and Yamauchi</span> |
<p>(1)insert: dddD</p> | <p>(1)insert: dddD</p> | ||
Line 2,311: | Line 2,385: | ||
<h4>Transformation</h4> | <h4>Transformation</h4> | ||
- | <span class= | + | <span class="kyoto-author">Yamauchi</span> |
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th><th colspan=2>CompetentCells/(µL)</th><th>Total/(µL)</th><th>Medium</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th colspan=2>CompetentCells/(µL)</th><th>Total/(µL)</th><th>Medium</th></tr> | ||
Line 2,333: | Line 2,407: | ||
<h4>Electrophoresis</h4> | <h4>Electrophoresis</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
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<tr><td>3</td><td>8/20 Ligation dddD + pSB1C3</td> | <tr><td>3</td><td>8/20 Ligation dddD + pSB1C3</td> | ||
<tr><td>4</td><td>8/1 Ligation dddD + pSB1C3</td> | <tr><td>4</td><td>8/1 Ligation dddD + pSB1C3</td> | ||
- | <tr><td>5</td><td>9/3 dddD (Gel extraction product)(E, | + | <tr><td>5</td><td>9/3 dddD (Gel extraction product)(E,P)</td> |
<tr><td>6</td><td>9/3 dddD (DNA purification product)</td> | <tr><td>6</td><td>9/3 dddD (DNA purification product)</td> | ||
<tr><td>7</td><td>1kb ladder</td> | <tr><td>7</td><td>1kb ladder</td> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/a/a1/Kyoto-dms-0904_electrophoresis_No1.jpg"> | ||
<!--"ゲノムを水?で戻す"を省略--> | <!--"ゲノムを水?で戻す"を省略--> | ||
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<h4>Cloning</h4> | <h4>Cloning</h4> | ||
- | <span class= | + | <span class="kyoto-author">Murata and Honda</span> |
<p>(1)</p> | <p>(1)</p> | ||
<table> | <table> | ||
Line 2,437: | Line 2,512: | ||
<th>Annealing</th> | <th>Annealing</th> | ||
<th>Extension</th> | <th>Extension</th> | ||
- | <th>Final | + | <th>Final Extension</th> |
<th>Cooling</th> | <th>Cooling</th> | ||
</tr> | </tr> | ||
Line 2,454: | Line 2,529: | ||
<td>90sec</td> | <td>90sec</td> | ||
<td>5min</td> | <td>5min</td> | ||
- | <td> | + | <td>Infinity</td> |
</tr> | </tr> | ||
</table> | </table> | ||
Line 2,466: | Line 2,541: | ||
<th>Annealing</th> | <th>Annealing</th> | ||
<th>Extension</th> | <th>Extension</th> | ||
- | <th>Final | + | <th>Final Extension</th> |
<th>Cooling</th> | <th>Cooling</th> | ||
</tr> | </tr> | ||
Line 2,483: | Line 2,558: | ||
<td>70sec</td> | <td>70sec</td> | ||
<td>5min</td> | <td>5min</td> | ||
- | <td> | + | <td>Infinity</td> |
</tr> | </tr> | ||
</table> | </table> | ||
Line 2,492: | Line 2,567: | ||
<h4>Electrophoresis</h4> | <h4>Electrophoresis</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
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<tr><td>6</td><td>1kb ladder</td> | <tr><td>6</td><td>1kb ladder</td> | ||
</table> | </table> | ||
- | + | <img src="https://static.igem.org/mediawiki/2014/d/db/Kyoto-dms-0904_cloning_electrophoresis_No1.jpg"> | |
<h4>Restriction Enzyme Digestion</h4> | <h4>Restriction Enzyme Digestion</h4> | ||
- | <span class= | + | <span class="kyoto-author">Yasuda</span> |
<p>(1)</p> | <p>(1)</p> | ||
<table> | <table> | ||
Line 2,542: | Line 2,617: | ||
<h4>Gel Extraction</h4> | <h4>Gel Extraction</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda and Yamauchi</span> |
<table> | <table> | ||
<tr><th>Lane</th><th>Sample</th></tr> | <tr><th>Lane</th><th>Sample</th></tr> | ||
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<tr><td>4</td><td>9/4 I732095(E,S)-1</td></tr> | <tr><td>4</td><td>9/4 I732095(E,S)-1</td></tr> | ||
<tr><td>5</td><td>-</td></tr> | <tr><td>5</td><td>-</td></tr> | ||
- | <tr><td>6</td><td>9/4 I732095(E,S)-1</td></tr> | + | <tr><td>6</td><td>9/4 I732095(E,S)-1 control</td></tr> |
</table> | </table> | ||
- | + | <img src="https://static.igem.org/mediawiki/2014/d/d3/Kyoto-dms-0905_Gel_Extraction_No1_before.jpg"> | |
+ | <img src="https://static.igem.org/mediawiki/2014/8/8b/Kyoto-dms-0905_Gel_Extraction_No1_after.jpg"> | ||
<h4>Genome Cloning</h4> | <h4>Genome Cloning</h4> | ||
Line 2,636: | Line 2,712: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td> | + | <td>5min</td> |
<td>20sec</td> | <td>20sec</td> | ||
<td>15sec</td> | <td>15sec</td> | ||
- | <td> | + | <td>90sec</td> |
- | <td> | + | <td>5min</td> |
- | <td> | + | <td>Infinity</td> |
</tr> | </tr> | ||
</table> | </table> | ||
Line 2,652: | Line 2,728: | ||
<h4>Electrophoresis</h4> | <h4>Electrophoresis</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<p>(1)AT</p> | <p>(1)AT</p> | ||
<table> | <table> | ||
Line 2,674: | Line 2,750: | ||
<tr><td>17</td><td>-</td></tr> | <tr><td>17</td><td>-</td></tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/b/bd/Kyoto-dms-0905_electrophoresis_AT.jpg"> | ||
<p>(2)SAMmt</p> | <p>(2)SAMmt</p> | ||
<table> | <table> | ||
Line 2,695: | Line 2,772: | ||
<tr><td>17</td><td>1kb ladder</td></tr> | <tr><td>17</td><td>1kb ladder</td></tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/2/2a/Kyoto-dms-0905_electrophoresis_SAMmt.jpg"> | ||
<p>(3)DiDECARB</p> | <p>(3)DiDECARB</p> | ||
<table> | <table> | ||
Line 2,716: | Line 2,794: | ||
<tr><td>17</td><td>-</td></tr> | <tr><td>17</td><td>-</td></tr> | ||
</table> | </table> | ||
- | + | <img src="https://static.igem.org/mediawiki/2014/8/8e/Kyoto-dms-0905_electrophoresis_DiDECARB.jpg"> | |
<h4>Gel Extraction</h4> | <h4>Gel Extraction</h4> | ||
- | <span class= | + | <span class="kyoto-author">Murata</span> |
<table> | <table> | ||
<tr><th>Lane</th><th>Sample</th></tr> | <tr><th>Lane</th><th>Sample</th></tr> | ||
Line 2,733: | Line 2,811: | ||
<tr><td>10</td><td>9/4 control</td></tr> | <tr><td>10</td><td>9/4 control</td></tr> | ||
</table> | </table> | ||
- | + | <img src="https://static.igem.org/mediawiki/2014/1/18/Kyoto-dms-0905_Gel_EXtraction_No2_before.jpg"> | |
+ | <img src="https://static.igem.org/mediawiki/2014/0/08/Kyoto-dms-0905_Gel_EXtraction_No2_after.jpg"> | ||
Line 2,829: | Line 2,908: | ||
<td>90sec</td> | <td>90sec</td> | ||
<td>5min</td> | <td>5min</td> | ||
- | <td> | + | <td>Infinity</td> |
</tr> | </tr> | ||
</table> | </table> | ||
Line 2,837: | Line 2,916: | ||
<h4>Electrophoresis</h4> | <h4>Electrophoresis</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
Line 2,847: | Line 2,926: | ||
<tr><td>6</td><td>1kb ladder</td></tr> | <tr><td>6</td><td>1kb ladder</td></tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/b/b2/Kyoto-dms-0908_electrophoresis_No1.jpg"> | ||
<h4>DNA Purification</h4> | <h4>DNA Purification</h4> | ||
- | <span class= | + | <span class="kyoto-author">Matsumoto</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
Line 2,862: | Line 2,942: | ||
<h4>Restriction Enzyme Digestion</h4> | <h4>Restriction Enzyme Digestion</h4> | ||
- | <span class= | + | <span class="kyoto-author">Matsumoto</span> |
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | ||
Line 2,914: | Line 2,994: | ||
<td>90sec</td> | <td>90sec</td> | ||
<td>5min</td> | <td>5min</td> | ||
- | <td> | + | <td>Infinity</td> |
</tr> | </tr> | ||
</table> | </table> | ||
Line 2,920: | Line 3,000: | ||
- | < | + | <!--パーツ起こしは書かない--> |
- | + | ||
- | + | ||
- | + | ||
Line 2,931: | Line 3,008: | ||
<h3>9/9</h3> | <h3>9/9</h3> | ||
- | <h4> | + | <h4>DNA purification</h4> |
- | <span class= | + | <span class="kyoto-author">Honda</span> |
+ | <table> | ||
+ | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>9/8 dddD(E,S)</td><td>1.3(x50)</td><td>1.66</td><td>1.28</td></tr> | ||
+ | </table> | ||
+ | |||
+ | |||
<h4>Ligation</h4> | <h4>Ligation</h4> | ||
- | <span class= | + | <span class="kyoto-author">Li</span> |
<table> | <table> | ||
- | (1)Sample | + | <p>(1)Sample</p> |
<tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | <tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | ||
<tr><td>pSB1C3</td><td>4.2</td><td>dddD</td><td>3.8</td><td>(2x)Buffer</td><td>9</td><td>T4 Ligase</td><td>1</td><td>-</td><td>18</td></tr> | <tr><td>pSB1C3</td><td>4.2</td><td>dddD</td><td>3.8</td><td>(2x)Buffer</td><td>9</td><td>T4 Ligase</td><td>1</td><td>-</td><td>18</td></tr> | ||
- | (2)Control | + | <p>(2)Control</p> |
<tr><td>pSB1C3</td><td>4.2</td><td>-</td><td>-</td><td>(2x)Buffer</td><td>9</td><td>T4 Ligase</td><td>1</td><td>3.8</td><td>18</td></tr> | <tr><td>pSB1C3</td><td>4.2</td><td>-</td><td>-</td><td>(2x)Buffer</td><td>9</td><td>T4 Ligase</td><td>1</td><td>3.8</td><td>18</td></tr> | ||
</table> | </table> | ||
Line 2,946: | Line 3,029: | ||
<h4>Transformation</h4> | <h4>Transformation</h4> | ||
- | <span class= | + | <span class="kyoto-author">Li</span> |
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th><th colspan=2>CompetentCells/(µL)</th><th>Total/(µL)</th><th>Medium</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th colspan=2>CompetentCells/(µL)</th><th>Total/(µL)</th><th>Medium</th></tr> | ||
Line 2,955: | Line 3,038: | ||
<h4>Liquid Culture</h4> | <h4>Liquid Culture</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Medium</th></tr> | <tr><th>Sample</th><th>Medium</th></tr> | ||
- | <tr><td> | + | <tr><td>9/8 T7-RBS</td><td>Plusgrow</td></tr> |
</table> | </table> | ||
<h4>PCR Cloning</h4> | <h4>PCR Cloning</h4> | ||
- | <span class= | + | <span class="kyoto-author">Murata</span> |
<table> | <table> | ||
Line 2,989: | Line 3,072: | ||
<th>Annealing</th> | <th>Annealing</th> | ||
<th>Extension</th> | <th>Extension</th> | ||
+ | <th>Final Extension</th> | ||
+ | <th>Cooling</th> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 3,003: | Line 3,088: | ||
<td>20sec</td> | <td>20sec</td> | ||
<td>15sec</td> | <td>15sec</td> | ||
- | <td> | + | <td>15sec</td> |
<td>5min</td> | <td>5min</td> | ||
- | <td> | + | <td>Infinity</td> |
</tr> | </tr> | ||
</table> | </table> | ||
Line 3,011: | Line 3,096: | ||
<h4>Gel Extraction</h4> | <h4>Gel Extraction</h4> | ||
- | <span class= | + | <span class="kyoto-author">Murata</span> |
<table> | <table> | ||
<tr><th>Lane</th><th>Sample</th></tr> | <tr><th>Lane</th><th>Sample</th></tr> | ||
Line 3,021: | Line 3,106: | ||
<tr><td>6</td><td>-</td></tr> | <tr><td>6</td><td>-</td></tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/4/41/Kyoto-dms-0909_Gel_Extraction_No1_after.jpg"> | ||
+ | |||
<h4>PCR</h4> | <h4>PCR</h4> | ||
Line 3,056: | Line 3,143: | ||
<td>98°C</td> | <td>98°C</td> | ||
<td>98°C</td> | <td>98°C</td> | ||
- | <td> | + | <td>63°C</td> |
<td>72°C</td> | <td>72°C</td> | ||
<td>72°C</td> | <td>72°C</td> | ||
Line 3,067: | Line 3,154: | ||
<td>90sec</td> | <td>90sec</td> | ||
<td>5min</td> | <td>5min</td> | ||
- | <td> | + | <td>Infinity</td> |
</tr> | </tr> | ||
</table> | </table> | ||
Line 3,082: | Line 3,169: | ||
<h4>Electrophoresis</h4> | <h4>Electrophoresis</h4> | ||
- | <span class= | + | <span class="kyoto-author">Murata</span> |
<table> | <table> | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
Line 3,094: | Line 3,181: | ||
<tr><td>8</td><td>-</td></tr> | <tr><td>8</td><td>-</td></tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/d/d3/Kyoto-dms-0910_electrophoresis_No1.jpg"> | ||
- | <h4>Miniprep | + | <h4>Miniprep</h4> |
- | <span class= | + | <span class="kyoto-author">Murata</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
Line 3,103: | Line 3,191: | ||
<h4>Cloning PCR</h4> | <h4>Cloning PCR</h4> | ||
- | <span class= | + | <span class="kyoto-author">Murata</span> |
<table> | <table> | ||
<tr> | <tr> | ||
- | < | + | <th>2x KAPA HiFi HotStart ReadyMix/(µL)</th> |
- | < | + | <th>Fw REDOX cloning primer 59/(µL)</th> |
- | < | + | <th>Rv REDOX primer Ver.2 64/(µL)</th> |
- | < | + | <th>DNA CCAP 1023/1 /(µL)</th> |
- | < | + | <th>MilliQ/(µL)</th> |
- | < | + | <th>Total/(µL)</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 3,125: | Line 3,213: | ||
<h4>Electrophoresis</h4> | <h4>Electrophoresis</h4> | ||
- | <span class= | + | <span class="kyoto-author">Murata</span> |
<table> | <table> | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
Line 3,137: | Line 3,225: | ||
<tr><td>8</td><td>-</td></tr> | <tr><td>8</td><td>-</td></tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/f/f7/Kyoto-dms-0910_electrophoresis_No2.jpg"> | ||
<h4>Colony PCR</h4> | <h4>Colony PCR</h4> | ||
Line 3,143: | Line 3,232: | ||
<table> | <table> | ||
<tr> | <tr> | ||
- | <th>Quick | + | <th>Quick Taq/(µL)</th> |
<th>VF2/(µL)</th> | <th>VF2/(µL)</th> | ||
<th>VR/(µL)</th> | <th>VR/(µL)</th> | ||
Line 3,217: | Line 3,306: | ||
</tr> | </tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/5/5b/Kyoto-dms-0910_colony_PCR_No1.jpg"> | ||
+ | |||
- | |||
- | |||
<table> | <table> | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
Line 3,226: | Line 3,315: | ||
<tr><td>3</td><td>-</td></tr> | <tr><td>3</td><td>-</td></tr> | ||
<tr><td>4</td><td>1kb ladder</td></tr> | <tr><td>4</td><td>1kb ladder</td></tr> | ||
- | <tr><td>5</td><td>9/9 dddD+ | + | <tr><td>5</td><td>9/9 dddD+pSB1C3 (1)</td></tr> |
- | <tr><td>6</td><td>9/9 dddD+ | + | <tr><td>6</td><td>9/9 dddD+pSB1C3 (2)</td></tr> |
- | <tr><td>7</td><td>9/9 dddD+ | + | <tr><td>7</td><td>9/9 dddD+pSB1C3 (3)</td></tr> |
- | <tr><td>8</td><td>9/9 dddD+ | + | <tr><td>8</td><td>9/9 dddD+pSB1C3 (4)</td></tr> |
<tr><td>9</td><td>1kb ladder</td></tr> | <tr><td>9</td><td>1kb ladder</td></tr> | ||
- | <tr><td>10</td><td>9/9 dddD+ | + | <tr><td>10</td><td>9/9 dddD+pSB1C3 (5)</td></tr> |
- | <tr><td>11</td><td>9/9 dddD+ | + | <tr><td>11</td><td>9/9 dddD+pSB1C3 (6)</td></tr> |
- | <tr><td>12</td><td>9/9 dddD+ | + | <tr><td>12</td><td>9/9 dddD+pSB1C3 (control)</td></tr> |
<tr><td>13</td><td>control</td></tr> | <tr><td>13</td><td>control</td></tr> | ||
<tr><td>14</td><td>1kb ladder</td></tr> | <tr><td>14</td><td>1kb ladder</td></tr> | ||
Line 3,240: | Line 3,329: | ||
<tr><td>17</td><td>-</td></tr> | <tr><td>17</td><td>-</td></tr> | ||
</table> | </table> | ||
- | |||
<h4>Electrophoresis</h4> | <h4>Electrophoresis</h4> | ||
- | <span class= | + | <span class="kyoto-author">Yasuda</span> |
<table> | <table> | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
Line 3,252: | Line 3,340: | ||
<tr><td>5</td><td>-</td></tr> | <tr><td>5</td><td>-</td></tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/e/ee/Kyoto-dms-0910_electrophoresis_No3.jpg"> | ||
- | <h4>Overlap | + | <h4>Overlap Extension(OE)-PCR</h4> |
- | <span class= | + | <span class="kyoto-author">Murata</span> |
<table> | <table> | ||
<tr> | <tr> | ||
- | < | + | <th>10x Buffer for KOD-Plus-Ver.2</th> |
- | < | + | <th>2mM dNTPs</th> |
- | < | + | <th>25mM MgSO4</th> |
- | < | + | <th>Primer(10µM each) #1</th> |
- | < | + | <th>Template DNA #2</th> |
- | < | + | <th>KOD-Plus-(1U/µL)</th> |
- | < | + | <th>MilliQ</th> |
- | < | + | <th>Total</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 3,321: | Line 3,410: | ||
<h4>Genome Cloning</h4> | <h4>Genome Cloning</h4> | ||
- | <span class= | + | <span class="kyoto-author">Yasuda</span> |
<table> | <table> | ||
<tr> | <tr> | ||
- | < | + | <th>2x KAPA HiFi HotStart ReadyMix/(µL)</th> |
- | < | + | <th>Fw REDOX cloning primer 59/(µL)</th> |
- | < | + | <th>Rv REDOX primer Ver.2 64/(µL)</th> |
- | < | + | <th>DNA CCAP 1023/1 /(µL)</th> |
- | < | + | <th>MilliQ/(µL)</th> |
- | < | + | <th>Total/(µL)</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 3,340: | Line 3,429: | ||
</tr> | </tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/2/24/Kyoto-dms-0910_genomic_cloning_No1.jpg"> | ||
<table> | <table> | ||
Line 3,356: | Line 3,446: | ||
<h4>Gel Extraction</h4> | <h4>Gel Extraction</h4> | ||
- | <span class= | + | <span class="kyoto-author">Yasuda</span> |
<p>(1)</p> | <p>(1)</p> | ||
<table> | <table> | ||
Line 3,373: | Line 3,463: | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/9/98/Kyoto-dms-0910_Gel_Extraction_No1_before.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/1/13/Kyoto-dms-0910_Gel_Extraction_No1_after.jpg"> | ||
- | (2) | + | <p>(2)</p> |
<table> | <table> | ||
<tr><th>Lane</th><th>Sample</th></tr> | <tr><th>Lane</th><th>Sample</th></tr> | ||
Line 3,385: | Line 3,477: | ||
<tr><td>7</td><td>1kb ladder</td></tr> | <tr><td>7</td><td>1kb ladder</td></tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/1/1f/Kyoto-dms-0910_Gel_Extraction_No2_before.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/1/19/Kyoto-dms-0910_Gel_Extraction_No2_after.jpg"> | ||
- | (3) | + | <p>(3)</p> |
<table> | <table> | ||
<tr><th>Lane</th><th>Sample</th></tr> | <tr><th>Lane</th><th>Sample</th></tr> | ||
Line 3,394: | Line 3,488: | ||
<tr><td>4</td><td>9/10 OE-PCR(6)</td></tr> | <tr><td>4</td><td>9/10 OE-PCR(6)</td></tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/8/8c/Kyoto-dms-0910_Gel_Extraction_No3_before.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/6/6b/Kyoto-dms-0910_Gel_Extraction_No3_after.jpg"> | ||
- | (4) | + | <p>(4)</p> |
<table> | <table> | ||
<tr><th>Lane</th><th>Sample</th></tr> | <tr><th>Lane</th><th>Sample</th></tr> | ||
Line 3,403: | Line 3,499: | ||
<tr><td>4</td><td>9/10 OE-PCR(7)</td></tr> | <tr><td>4</td><td>9/10 OE-PCR(7)</td></tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/d/d6/Kyoto-dms-0910_Gel_Extraction_No4_before.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/9/9b/Kyoto-dms-0910_Gel_Extraction_No4_after.jpg"> | ||
- | (5) | + | <p>(5)</p> |
<table> | <table> | ||
<tr><th>Lane</th><th>Sample</th></tr> | <tr><th>Lane</th><th>Sample</th></tr> | ||
Line 3,412: | Line 3,510: | ||
<tr><td>4</td><td>9/10 OE-PCR(8)</td></tr> | <tr><td>4</td><td>9/10 OE-PCR(8)</td></tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/7/7f/Kyoto-dms-0910_Gel_Extraction_No5_before.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/3/31/Kyoto-dms-0910_Gel_Extraction_No5_after.jpg"> | ||
- | (6) | + | <p>(6)</p> |
<table> | <table> | ||
<tr><th>Lane</th><th>Sample</th></tr> | <tr><th>Lane</th><th>Sample</th></tr> | ||
Line 3,423: | Line 3,523: | ||
<tr><td>6</td><td>9/10 OE-PCR(10)</td></tr> | <tr><td>6</td><td>9/10 OE-PCR(10)</td></tr> | ||
</table> | </table> | ||
- | + | <img src="https://static.igem.org/mediawiki/2014/a/ae/Kyoto-dms-0910_Gel_Extraction_No6_before.jpg"> | |
- | + | <img src="https://static.igem.org/mediawiki/2014/5/54/Kyoto-dms-0910_Gel_Extraction_No6_after.jpg"> | |
<h4>Ligation</h4> | <h4>Ligation</h4> | ||
- | <span class= | + | <span class="kyoto-author">Tatsui and Murata</span> |
<table> | <table> | ||
<tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | <tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | ||
Line 3,434: | Line 3,534: | ||
<h4>Liquid Culture</h4> | <h4>Liquid Culture</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Medium</th></tr> | <tr><th>Sample</th><th>Medium</th></tr> | ||
- | <tr><td>9/9 dddD+ | + | <tr><td>9/9 dddD+pSB1C3-(1)</td><td>CP</td></tr> |
</table> | </table> | ||
<h4>Restriction Enzyme Digestion</h4> | <h4>Restriction Enzyme Digestion</h4> | ||
- | <span class= | + | <span class="kyoto-author">Shimizu</span> |
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | ||
Line 3,453: | Line 3,553: | ||
<h4>OE-PCR</h4> | <h4>OE-PCR</h4> | ||
- | <span class= | + | <span class="kyoto-author">Shimizu</span> |
<table> | <table> | ||
<tr> | <tr> | ||
- | < | + | <th>10x Buffer for KOD-Plus-Ver.2</th> |
- | < | + | <th>2mM dNTPs</th> |
- | < | + | <th>25mM MgSO4</th> |
- | < | + | <th>Primer(10µM each) #1</th> |
- | < | + | <th>Template DNA #2</th> |
- | < | + | <th>KOD-Plus-(1U/µL)</th> |
- | < | + | <th>MilliQ</td> |
- | < | + | <th>Total</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 3,498: | Line 3,598: | ||
<th>Annealing</th> | <th>Annealing</th> | ||
<th>Extension</th> | <th>Extension</th> | ||
- | <th>Last | + | <th>Last Extension</th> |
<th>Cooling</th> | <th>Cooling</th> | ||
</tr> | </tr> | ||
Line 3,515: | Line 3,615: | ||
<td>2min10sec</td> | <td>2min10sec</td> | ||
<td>5min</td> | <td>5min</td> | ||
- | <td> | + | <td>Infinity</td> |
</tr> | </tr> | ||
</table> | </table> | ||
Line 3,524: | Line 3,624: | ||
<h4>Ligation</h4> | <h4>Ligation</h4> | ||
- | <span class= | + | <span class="kyoto-author">Shimizu</span> |
<table> | <table> | ||
<tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | <tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | ||
Line 3,545: | Line 3,645: | ||
<h4>Miniprep</h4> | <h4>Miniprep</h4> | ||
- | <span class= | + | <span class="kyoto-author">Murata</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
Line 3,552: | Line 3,652: | ||
<h4>DNA Purification</h4> | <h4>DNA Purification</h4> | ||
- | <span class= | + | <span class="kyoto-author">Matsumoto</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
Line 3,561: | Line 3,661: | ||
<h4>OE-PCR</h4> | <h4>OE-PCR</h4> | ||
- | <span class= | + | <span class="kyoto-author">Shimizu</span> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 3,625: | Line 3,725: | ||
<h4>Electrophoresis</h4> | <h4>Electrophoresis</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda and Murata</span> |
<table> | <table> | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
Line 3,636: | Line 3,736: | ||
<tr><td>7</td><td>1kb ladder</td></tr> | <tr><td>7</td><td>1kb ladder</td></tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/c/ca/Kyoto-dms-0911_electrophoresis_No1.jpg"> | ||
<h4>Colony PCR</h4> | <h4>Colony PCR</h4> | ||
- | <span class= | + | <span class="kyoto-author">Murata and Yamauchi</span> |
<p>Master Mix (x32)</p> | <p>Master Mix (x32)</p> | ||
Line 3,744: | Line 3,845: | ||
<h4>Mutation PCR</h4> | <h4>Mutation PCR</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<p>(1)dddD (PstI)</p> | <p>(1)dddD (PstI)</p> | ||
<table> | <table> | ||
<tr> | <tr> | ||
- | < | + | <th>Prime STAR MAX Premix(2x)</th> |
- | < | + | <th>Fw dddD PstI mutation primer</th> |
- | < | + | <th>Rv dddD PstI mutation primer</th> |
- | < | + | <th>9/11 MP dddD (x2000)</th> |
- | < | + | <th>MilliQ</th> |
- | < | + | <th>Total</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 3,769: | Line 3,870: | ||
<table> | <table> | ||
<tr> | <tr> | ||
- | < | + | <th>Prime STAR MAX Premix(2x)</th> |
- | < | + | <th>Fw pSB1C3-his-F-v2 primer</th> |
- | < | + | <th>Rv pSB1C3 Histag mutation primer</th> |
- | < | + | <th>9/10 MP T7-RBS (x3000)</th> |
- | < | + | <th>MilliQ</th> |
- | < | + | <th>Total</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 3,790: | Line 3,891: | ||
<table> | <table> | ||
<tr> | <tr> | ||
- | < | + | <th>Prime STAR MAX Premix(2x)</th> |
- | < | + | <th>Fw Control primer</th> |
- | < | + | <th>Rv Control primer</th> |
- | < | + | <th>Control template (x1000)</th> |
- | < | + | <th>MilliQ</td> |
- | < | + | <th>Total</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 3,813: | Line 3,914: | ||
<th>Annealing</th> | <th>Annealing</th> | ||
<th>Extension</th> | <th>Extension</th> | ||
- | |||
<th>Cooling</th> | <th>Cooling</th> | ||
</tr> | </tr> | ||
Line 3,820: | Line 3,920: | ||
<td>98°C</td> | <td>98°C</td> | ||
<td>55°C</td> | <td>55°C</td> | ||
- | |||
<td>72°C</td> | <td>72°C</td> | ||
<td>12°C</td> | <td>12°C</td> | ||
Line 3,829: | Line 3,928: | ||
<td>15sec</td> | <td>15sec</td> | ||
<td>25sec</td> | <td>25sec</td> | ||
- | <td> | + | <td>Infinity</td> |
- | + | ||
</tr> | </tr> | ||
</table> | </table> | ||
Line 3,838: | Line 3,936: | ||
<h4>DNA Purification</h4> | <h4>DNA Purification</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
Line 3,845: | Line 3,943: | ||
<h4>Gel Extraction</h4> | <h4>Gel Extraction</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<p>(1)</p> | <p>(1)</p> | ||
<table> | <table> | ||
Line 3,861: | Line 3,959: | ||
<tr><td>11</td><td>9/11 OE-PCR-(3)</td></tr> | <tr><td>11</td><td>9/11 OE-PCR-(3)</td></tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/0/0b/Kyoto-dms-0911_Gel_Extraction_No1_before.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/c/c8/Kyoto-dms-0911_Gel_Extraction_No1_after.jpg"> | ||
<p>(2)</p> | <p>(2)</p> | ||
Line 3,877: | Line 3,977: | ||
<tr><td>11</td><td>9/11 OE-PCR-(10)</td></tr> | <tr><td>11</td><td>9/11 OE-PCR-(10)</td></tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/9/90/Kyoto-dms-0911_Gel_Extraction_No2_before.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/9/96/Kyoto-dms-0911_Gel_Extraction_No2_after.jpg"> | ||
<p>(3)</p> | <p>(3)</p> | ||
Line 3,893: | Line 3,995: | ||
<tr><td>11</td><td>9/11 OE-PCR-(16)</td></tr> | <tr><td>11</td><td>9/11 OE-PCR-(16)</td></tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/f/fd/Kyoto-dms-0911_Gel_Extraction_No3_before.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/0/0f/Kyoto-dms-0911_Gel_Extraction_No3_after.jpg"> | ||
- | + | <h4>Gel Extraction</h4> | |
+ | <span class="kyoto-author">Li</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>(1)</td> | ||
+ | <td>0.1</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>(2)</td> | ||
+ | <td>4.9</td> | ||
+ | <td>1.67</td> | ||
+ | <td>1.95</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>(3)</td> | ||
+ | <td>4.6</td> | ||
+ | <td>1.74</td> | ||
+ | <td>1.24</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>(6)</td> | ||
+ | <td>6.3</td> | ||
+ | <td>1.61</td> | ||
+ | <td>2.03</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>(8)</td> | ||
+ | <td>2.6</td> | ||
+ | <td>1.19</td> | ||
+ | <td>0.95</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>(10)</td> | ||
+ | <td>8.5</td> | ||
+ | <td>1.52</td> | ||
+ | <td>1.37</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>(12)</td> | ||
+ | <td>1.8</td> | ||
+ | <td>1.44</td> | ||
+ | <td>1.51</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>(14)</td> | ||
+ | <td>2.4</td> | ||
+ | <td>1.85</td> | ||
+ | <td>0.82</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>(16)</td> | ||
+ | <td>5.0</td> | ||
+ | <td>1.72</td> | ||
+ | <td>2.21</td> | ||
+ | </tr> | ||
+ | </table> | ||
<h4>DNA Purification</h4> | <h4>DNA Purification</h4> | ||
- | <span class= | + | <span class="kyoto-author">Yamauchi</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
Line 3,919: | Line 4,086: | ||
<h4>Ligation</h4> | <h4>Ligation</h4> | ||
- | <span class= | + | <span class="kyoto-author">Tatsui and Yamauchi</span> |
<table> | <table> | ||
<tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | <tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | ||
Line 3,925: | Line 4,092: | ||
<tr><td>YC plac111 (SmaI cut)</td><td>1</td><td>9/11 SAMmt (DNA purification product)</td><td>0.5</td><td>(2x) Buufer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>2.5</td><td>9</td></tr> | <tr><td>YC plac111 (SmaI cut)</td><td>1</td><td>9/11 SAMmt (DNA purification product)</td><td>0.5</td><td>(2x) Buufer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>2.5</td><td>9</td></tr> | ||
- | |||
<tr><td>YC plac111 (SmaI cut)</td><td>1</td><td>9/11 DiDECARB (DNA purification product)</td><td>1</td><td>(2x) Buufer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>2</td><td>9</td></tr> | <tr><td>YC plac111 (SmaI cut)</td><td>1</td><td>9/11 DiDECARB (DNA purification product)</td><td>1</td><td>(2x) Buufer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>2</td><td>9</td></tr> | ||
Line 3,945: | Line 4,111: | ||
<h4>OE-PCR</h4> | <h4>OE-PCR</h4> | ||
- | <span class= | + | <span class="kyoto-author">Yasuda</span> |
<table> | <table> | ||
<tr> | <tr> | ||
- | < | + | <th>10x Buffer for KOD-Plus-Ver.2</th> |
- | < | + | <th>2mM dNTPs</th> |
- | < | + | <th>25mM MgSO4</th> |
- | < | + | <th>Primer(10µM each) #1</th> |
- | < | + | <th>Template DNA #2</th> |
- | < | + | <th>KOD-Plus-(1U/µL)</th> |
- | < | + | <th>MilliQ</th> |
- | < | + | <th>Total</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 3,989: | Line 4,155: | ||
<th>Annealing</th> | <th>Annealing</th> | ||
<th>Extension</th> | <th>Extension</th> | ||
- | <th>Last | + | <th>Last Extension</th> |
<th>Cooling</th> | <th>Cooling</th> | ||
</tr> | </tr> | ||
Line 4,006: | Line 4,172: | ||
<td>1min</td> | <td>1min</td> | ||
<td>5min</td> | <td>5min</td> | ||
- | <td> | + | <td>Infinity</td> |
</tr> | </tr> | ||
</table> | </table> | ||
Line 4,020: | Line 4,186: | ||
<h4>Electrophoresis</h4> | <h4>Electrophoresis</h4> | ||
- | <span class= | + | <span class="kyoto-author">Li</span> |
<table> | <table> | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
- | <tr><td>1</td><td>1kb ladder</td></tr> | + | <tr><td>1</td><td>Blank</td></tr> |
- | <tr><td> | + | <tr><td>2</td><td>1kb ladder</td></tr> |
- | <tr><td> | + | <tr><td>3</td><td>9/11 OE-PCR 2nd (1)</td></tr> |
- | <tr><td> | + | <tr><td>4</td><td>9/11 OE-PCR 2nd (2)</td></tr> |
- | <tr><td> | + | <tr><td>5</td><td>9/11 OE-PCR 2nd (3)</td></tr> |
+ | <tr><td>6</td><td>9/11 OE-PCR 2nd (4)</td></tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/3/36/Kyoto-dms-0912_DNA_purification_No1.jpg"> | ||
<h4>DNA Purification</h4> | <h4>DNA Purification</h4> | ||
- | <span class= | + | <span class="kyoto-author">Li</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
Line 4,042: | Line 4,210: | ||
<h4>Transformation</h4> | <h4>Transformation</h4> | ||
- | <span class= | + | <span class="kyoto-author">Yamauchi</span> |
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th><th colspan=2>CompetentCells/(µL)</th><th>Total/(µL)</th><th>Medium</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th colspan=2>CompetentCells/(µL)</th><th>Total/(µL)</th><th>Medium</th></tr> | ||
- | <tr><td>9/11 SAMmt (ligation product)</td><td>2</td><td>DH5& | + | <tr><td>9/11 SAMmt (ligation product)</td><td>2</td><td>DH5α</td><td>10</td><td>12</td><td>CP</td></tr> |
- | <tr><td>9/11 DiDECARB (ligation product)</td><td>2</td><td>DH5& | + | <tr><td>9/11 DiDECARB (ligation product)</td><td>2</td><td>DH5α</td><td>10</td><td>12</td><td>CP</td></tr> |
- | <tr><td>9/11 DECARB (ligation product)</td><td>2</td><td>DH5& | + | <tr><td>9/11 DECARB (ligation product)</td><td>2</td><td>DH5α</td><td>10</td><td>12</td><td>CP</td></tr> |
- | <tr><td>9/11 REDOX (ligation product)</td><td>2</td><td>DH5& | + | <tr><td>9/11 REDOX (ligation product)</td><td>2</td><td>DH5α</td><td>10</td><td>12</td><td>CP</td></tr> |
</table> | </table> | ||
- | <h4>Sequence | + | <h4>Sequence</h4> |
- | <span class= | + | <span class="kyoto-author">Yasuda</span> |
<table> | <table> | ||
<tr><th>Bis Dye v3.1 Sequencing RR-24(/µL)</th> | <tr><th>Bis Dye v3.1 Sequencing RR-24(/µL)</th> | ||
Line 4,074: | Line 4,242: | ||
<p> | <p> | ||
- | 1. VF | + | 1. VF<br> |
2. Sequence No.1<br> | 2. Sequence No.1<br> | ||
3. Sequence No.2<br> | 3. Sequence No.2<br> | ||
Line 4,082: | Line 4,250: | ||
7. Sequence No.6<br> | 7. Sequence No.6<br> | ||
</p> | </p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
<h4>Liquid Culture</h4> | <h4>Liquid Culture</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Medium</th></tr> | <tr><th>Sample</th><th>Medium</th></tr> | ||
- | <tr><td>9/10 dddD-YCplac111 (Transformation product)</td><td> | + | <tr><td>9/10 dddD-YCplac111 (Transformation product)</td><td>Amp</td></tr> |
</table> | </table> | ||
Line 4,092: | Line 4,264: | ||
<h4>Restriction Enzyme Digestion</h4> | <h4>Restriction Enzyme Digestion</h4> | ||
- | <span class= | + | <span class="kyoto-author">Murata</span> |
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | ||
Line 4,170: | Line 4,342: | ||
<td>72°C</td> | <td>72°C</td> | ||
<td>72°C</td> | <td>72°C</td> | ||
- | <td> | + | <td>4°C</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 4,178: | Line 4,350: | ||
<td>90sec</td> | <td>90sec</td> | ||
<td>5min</td> | <td>5min</td> | ||
- | <td> | + | <td>Infinity</td> |
</tr> | </tr> | ||
</table> | </table> | ||
Line 4,185: | Line 4,357: | ||
<h4>Gel Extraction</h4> | <h4>Gel Extraction</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th>Lane</th><th>Sample</th></tr> | <tr><th>Lane</th><th>Sample</th></tr> | ||
Line 4,195: | Line 4,367: | ||
<tr><td>6</td><td>1kb ladder</td></tr> | <tr><td>6</td><td>1kb ladder</td></tr> | ||
</table> | </table> | ||
- | + | <img src="https://static.igem.org/mediawiki/2014/6/64/Kyoto-dms-0912_Gel_Extention_No1_before.jpg"> | |
+ | <img src="https://static.igem.org/mediawiki/2014/9/98/Kyoto-dms-0912_Gel_Extention_No1_after.jpg"> | ||
<h4>Cloning PCR (for Histag)</h4> | <h4>Cloning PCR (for Histag)</h4> | ||
- | <span class= | + | <span class="kyoto-author">Murata</span> |
<table> | <table> | ||
<tr> | <tr> | ||
- | < | + | <th>2x KAPA HiFi HotStart ReadyMix/(µL)</th> |
- | < | + | <th>Fw 9/10 AT OE-A/(µL)</th> |
- | < | + | <th>Rv 9/12 AT His/(µL)</th> |
- | < | + | <th>Templete 9/11 OE-PCR 2nd (1) 9/12/(µL)</th> |
- | < | + | <th>MilliQ/(µL)</th> |
- | < | + | <th>Total/(µL)</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 4,242: | Line 4,415: | ||
<td>45sec</td> | <td>45sec</td> | ||
<td>5min</td> | <td>5min</td> | ||
- | <td> | + | <td>Infinity</td> |
</tr> | </tr> | ||
</table> | </table> | ||
Line 4,250: | Line 4,423: | ||
<h4>Miniprep</h4> | <h4>Miniprep</h4> | ||
- | <span class= | + | <span class="kyoto-author">Tatsui</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
- | <tr><td>dddD- | + | <tr><td>dddD-pSB1C3 (Liquid culture product)</td><td>370</td><td>1.75</td><td>2.48</td></tr> |
</table> | </table> | ||
<h4>DNA Purification</h4> | <h4>DNA Purification</h4> | ||
- | <span class= | + | <span class="kyoto-author">Tatsui</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
Line 4,268: | Line 4,441: | ||
<h4>Restriction Enzyme Digestion</h4> | <h4>Restriction Enzyme Digestion</h4> | ||
- | <span class= | + | <span class="kyoto-author">Tatsui</span> |
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | ||
Line 4,277: | Line 4,450: | ||
<h4>PCR (for Control)</h4> | <h4>PCR (for Control)</h4> | ||
- | <span class= | + | <span class="kyoto-author">Murata</span> |
<p>(1)</p> | <p>(1)</p> | ||
<table> | <table> | ||
Line 4,374: | Line 4,547: | ||
<td>95°C</td> | <td>95°C</td> | ||
<td>98°C</td> | <td>98°C</td> | ||
- | <td> | + | <td>60°C</td> |
<td>72°C</td> | <td>72°C</td> | ||
<td>72°C</td> | <td>72°C</td> | ||
- | <td> | + | <td>4°C</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 4,383: | Line 4,556: | ||
<td>20sec</td> | <td>20sec</td> | ||
<td>15sec</td> | <td>15sec</td> | ||
- | <td> | + | <td>90sec</td> |
<td>5min</td> | <td>5min</td> | ||
- | <td> | + | <td>Infinity</td> |
</tr> | </tr> | ||
</table> | </table> | ||
Line 4,394: | Line 4,567: | ||
<h4>Restriction Enzyme Digestion</h4> | <h4>Restriction Enzyme Digestion</h4> | ||
- | <span class= | + | <span class="kyoto-author">Yamauchi</span> |
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | ||
Line 4,407: | Line 4,580: | ||
<h4>Ligation</h4> | <h4>Ligation</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | <tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | ||
- | <tr><td>YC plac111</td> | + | <tr><td>YC plac111</td><td>1</td><td>AT-His cloning</td><td>0.5</td><td>(2x)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>3.5</td><td>10</td></tr> |
- | <tr><td>YC plac111</td> | + | <tr><td>YC plac111</td><td>1</td><td>AT</td><td>0.5</td><td>(2x)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>3.5</td><td>10</td></tr> |
- | <tr><td> | + | <tr><td>pSB1C3 (E,P)</td><td>1.5</td><td>SAMmt (E,P)</td><td>1</td><td>(2x)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>2.5</td><td>10</td></tr> |
</table> | </table> | ||
Line 4,421: | Line 4,594: | ||
<h4>DNA Purification</h4> | <h4>DNA Purification</h4> | ||
- | <span class= | + | <span class="kyoto-author">Tatsui</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
Line 4,433: | Line 4,606: | ||
<h4>Electrophoresis</h4> | <h4>Electrophoresis</h4> | ||
- | <span class= | + | <span class="kyoto-author"></span> |
<table> | <table> | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
Line 4,453: | Line 4,626: | ||
<tr><td>16</td><td>-</td></tr> | <tr><td>16</td><td>-</td></tr> | ||
</table> | </table> | ||
- | + | <img src="https://static.igem.org/mediawiki/2014/7/74/Kyoto-dms-0912_Elecrophoresis_No1.jpg"> | |
<h4>Transformation</h4> | <h4>Transformation</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th><th colspan=2>CompetentCells/(µL)</th><th>Total/(µL)</th><th>Medium</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th colspan=2>CompetentCells/(µL)</th><th>Total/(µL)</th><th>Medium</th></tr> | ||
Line 4,464: | Line 4,637: | ||
<tr><td>AT-YCplac111</td><td>1</td><td>DH5α</td><td>10</td><td>11</td><td>SOD</td></tr> | <tr><td>AT-YCplac111</td><td>1</td><td>DH5α</td><td>10</td><td>11</td><td>SOD</td></tr> | ||
- | <tr><td>SAMmt(E,P)- | + | <tr><td>SAMmt(E,P)-pSB1C3(E,P)</td><td>1</td><td>DH5α</td><td>10</td><td>11</td><td>SOD</td></tr> |
</table> | </table> | ||
<h4>Transformation</h4> | <h4>Transformation</h4> | ||
- | <span class= | + | <span class="kyoto-author">Yasuda</span> |
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th><th colspan=2>CompetentCells/(µL)</th><th>Total/(µL)</th><th>Medium</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th colspan=2>CompetentCells/(µL)</th><th>Total/(µL)</th><th>Medium</th></tr> | ||
Line 4,480: | Line 4,653: | ||
- | <h4>Colony PCR | + | <h4>Colony PCR</h4> |
<span class="kyoto-auther">Tatsui and Li</span> | <span class="kyoto-auther">Tatsui and Li</span> | ||
+ | <br> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/7/7f/Kyoto-dms-0912_Colony_PCR_No1.jpg"> | ||
- | |||
- | |||
<table> | <table> | ||
- | <tr><th> | + | <tr> |
- | < | + | <th>Predenature</th> |
- | < | + | <th>Denature</th> |
- | < | + | <th>Annealing</th> |
- | < | + | <th>Extension</th> |
- | + | <th>Last Extension</th> | |
- | < | + | <th>Cooling</th> |
- | + | </tr> | |
- | + | <tr> | |
- | + | <td>94°C</td> | |
- | + | <td>94°C</td> | |
- | + | <td>55°C</td> | |
- | + | <td>68°C</td> | |
- | <tr><td> | + | <td>68°C</td> |
- | + | <td>12°C</td> | |
- | + | </tr> | |
- | + | <tr> | |
+ | <td>2min</td> | ||
+ | <td>30sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>1min</td> | ||
+ | <td>1sec</td> | ||
+ | <td>Infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 25cycle</p> | ||
+ | <table> | ||
+ | <tr><th>Lane</th><th>Sample</th></tr> | ||
+ | <tr><td>1-8</td><td>YCplac111-SAMmt (1)-(8)</td></tr> | ||
+ | <tr><td>9</td><td>1kb ladder</td></tr> | ||
+ | <tr><td>10-16</td><td>YCplac111-REDOX (1)-(7)</td></tr> | ||
</table> | </table> | ||
- | <h4>Liquid Culture | + | |
- | <span class= | + | <h4>Liquid Culture</h4> |
+ | <span class="kyoto-author">Yamauchi</span> | ||
<table> | <table> | ||
<tr><th>Sample</th><th>Medium</th></tr> | <tr><th>Sample</th><th>Medium</th></tr> | ||
- | <tr><td>9/5 Pcon- | + | <tr><td>9/5 Pcon-pSB1C3-(3)</td><td>CP</td></tr> |
- | <tr><td>9/5 Pcon- | + | <tr><td>9/5 Pcon-pSB1C3-(3)</td><td>CP</td></tr> |
- | <tr><td>9/11 SAMmt-(3)</td><td></td> | + | <tr><td>9/11 SAMmt-(3)</td><td>Amp</td></tr> |
- | <tr><td>9/8 T7-RBS-(1)</td><td></td> | + | <tr><td>9/8 T7-RBS-(1)</td><td>CP</td></tr> |
- | <tr><td>9/8 T7-RBS-(1)</td><td></td> | + | <tr><td>9/8 T7-RBS-(1)</td><td>CP</td></tr> |
</table> | </table> | ||
Line 4,526: | Line 4,714: | ||
<table> | <table> | ||
<tr> | <tr> | ||
- | <th>Quick | + | <th>Quick Taq/(µL)</th> |
<th>M13 Fw/(µL)</th> | <th>M13 Fw/(µL)</th> | ||
<th>M13 Rv/(µL)</th> | <th>M13 Rv/(µL)</th> | ||
Line 4,540: | Line 4,728: | ||
</tr> | </tr> | ||
</table> | </table> | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
<table> | <table> | ||
<tr> | <tr> | ||
Line 4,555: | Line 4,735: | ||
<th>Annealing</th> | <th>Annealing</th> | ||
<th>Extension</th> | <th>Extension</th> | ||
- | <th>Last | + | <th>Last Extension</th> |
<th>Cooling</th> | <th>Cooling</th> | ||
</tr> | </tr> | ||
Line 4,571: | Line 4,751: | ||
<td>30sec</td> | <td>30sec</td> | ||
<td>1min</td> | <td>1min</td> | ||
- | <td> | + | <td>1min</td> |
- | <td> | + | <td>Infinity</td> |
</tr> | </tr> | ||
</table> | </table> | ||
- | <p>Denature-Annealing-Extension | + | <p>Denature-Annealing-Extension 30cycle</p> |
+ | <img src="https://static.igem.org/mediawiki/2014/5/51/Kyoto-dms-0912_Colony_PCR_No2.jpg"> | ||
- | |||
- | |||
- | |||
<table> | <table> | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
- | <tr><td>1 | + | <tr><td>1-8</td><td>written in "magnetosome"</td></tr> |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
<tr><td>9</td><td>1kb ladder</td></tr> | <tr><td>9</td><td>1kb ladder</td></tr> | ||
- | <tr><td>10</td><td>REDOX-YCplac111 | + | <tr><td>10-17</td><td>REDOX-YCplac111 (1)-(8)</td></tr> |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
</table> | </table> | ||
+ | |||
+ | |||
+ | |||
Line 4,615: | Line 4,783: | ||
<h4>Miniprep</h4> | <h4>Miniprep</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
- | <tr><td>9/12 T7-RBS- | + | <tr><td>9/12 T7-RBS-pSB1C3 (liquid culture product)</td><td>95</td><td>2.02</td><td>2.72</td></tr> |
<tr><td>9/12 SAMmt-YCplac111 (liquid culture product)</td><td>110</td><td>1.70</td><td>1.15</td></tr> | <tr><td>9/12 SAMmt-YCplac111 (liquid culture product)</td><td>110</td><td>1.70</td><td>1.15</td></tr> | ||
Line 4,628: | Line 4,796: | ||
<h4>Gel Extraction</h4> | <h4>Gel Extraction</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda and Yasuda</span> |
<table> | <table> | ||
<tr><th>Lane</th><th>Sample</th></tr> | <tr><th>Lane</th><th>Sample</th></tr> | ||
Line 4,634: | Line 4,802: | ||
<tr><td>2</td><td>-</td></tr> | <tr><td>2</td><td>-</td></tr> | ||
<tr><td>3</td><td>-</td></tr> | <tr><td>3</td><td>-</td></tr> | ||
- | <tr><td>4</td><td> | + | <tr><td>4</td><td>pSB1C3-Pcon(X,S)</td></tr> |
<tr><td>5</td><td>-</td></tr> | <tr><td>5</td><td>-</td></tr> | ||
- | <tr><td>6</td><td>Control ( | + | <tr><td>6</td><td>Control (pSB1C3-Pcon(X,S))</td></tr> |
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/c/cc/Kyoto-dms-0913_Gel_Extention_No1_before.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/c/c3/Kyoto-dms-0913_Gel_Extention_No1_after.jpg"> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pSB1C3-Pcon X,S</td> | ||
+ | <td>1.5</td> | ||
+ | <td>1.11</td> | ||
+ | <td>0.59</td> | ||
+ | </tr> | ||
+ | </table> | ||
- | <h4> | + | <h4>DNA Purification</h4> |
- | <span class= | + | <span class="kyoto-author">Yasuda</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
Line 4,652: | Line 4,836: | ||
<h4>Ligation</h4> | <h4>Ligation</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | <tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | ||
- | <tr><td>9/8 | + | <tr><td>9/8 pSB1C3(E,P)</td><td>3</td><td>9/13 AT(E,P)(DNA purification product)</td><td>1</td><td>(2x)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>0</td><td>10</td></tr> |
- | <tr><td>9/13 | + | <tr><td>9/13 pSB1C3(X,P)</td><td>3</td><td>9/13 AT-His(X,P)(DNA purification product)</td><td>1</td><td>(2x)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>0</td><td>10</td></tr> |
</table> | </table> | ||
<h4>Miniprep</h4> | <h4>Miniprep</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
<tr><td>9/12 SAMmt-YCplac111</td><td>140</td><td>1.80</td><td>1.90</td></tr> | <tr><td>9/12 SAMmt-YCplac111</td><td>140</td><td>1.80</td><td>1.90</td></tr> | ||
- | <tr><td>T7-RBS- | + | <tr><td>T7-RBS-pSB1C3</td><td>290</td><td>1.78</td><td>1.65</td></tr> |
</table> | </table> | ||
<h4>Liquid Culture</h4> | <h4>Liquid Culture</h4> | ||
- | <span class= | + | <span class="kyoto-author">Yasuda</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Medium</th></tr> | <tr><th>Sample</th><th>Medium</th></tr> | ||
- | <tr><td>9/12 dddD- | + | <tr><td>9/12 dddD-pSB1C3-(1)</td><td>CP</td></tr> |
- | <tr><td>9/12 dddD- | + | <tr><td>9/12 dddD-pSB1C3-(2)</td><td>CP</td></tr> |
- | <tr><td>9/12 dddD- | + | <tr><td>9/12 dddD-pSB1C3-(3)</td><td>CP</td></tr> |
<tr><td>9/12 T7-RBS-His-(1)</td><td>CP</td></tr> | <tr><td>9/12 T7-RBS-His-(1)</td><td>CP</td></tr> | ||
<tr><td>9/12 T7-RBS-His-(2)</td><td>CP</td></tr> | <tr><td>9/12 T7-RBS-His-(2)</td><td>CP</td></tr> | ||
Line 4,696: | Line 4,880: | ||
<h4>Colony PCR</h4> | <h4>Colony PCR</h4> | ||
- | <span class= | + | <span class="kyoto-author">Yasuda</span> |
Line 4,721: | Line 4,905: | ||
<th>Annealing</th> | <th>Annealing</th> | ||
<th>Extension</th> | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 4,727: | Line 4,912: | ||
<td>55°C</td> | <td>55°C</td> | ||
<td>68°C</td> | <td>68°C</td> | ||
+ | <td>12°C</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 4,733: | Line 4,919: | ||
<td>30sec</td> | <td>30sec</td> | ||
<td>160sec</td> | <td>160sec</td> | ||
+ | <td>infinity</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
Line 4,748: | Line 4,935: | ||
<h4>Gel Extraction</h4> | <h4>Gel Extraction</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th>Lane</th><th>Sample</th></tr> | <tr><th>Lane</th><th>Sample</th></tr> | ||
Line 4,755: | Line 4,942: | ||
<tr><td>3</td><td>9/13 SAMmt (E,P)</td></tr> | <tr><td>3</td><td>9/13 SAMmt (E,P)</td></tr> | ||
<tr><td>4</td><td>-</td></tr> | <tr><td>4</td><td>-</td></tr> | ||
- | <tr><td>5</td><td>9/13 Control (Pcon- | + | <tr><td>5</td><td>9/13 Control (Pcon-pSB1C3)</td></tr> |
- | <tr><td>6</td><td>9/13 Pcon- | + | <tr><td>6</td><td>9/13 Pcon-pSB1C3(X,S)</td></tr> |
</table> | </table> | ||
- | + | <img src="https://static.igem.org/mediawiki/2014/d/d3/Kyoto-dms-0914_Gel_Extention_No1_before%26after.jpg"> | |
<h4>DNA Purification</h4> | <h4>DNA Purification</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
- | <tr><td>Pcon- | + | <tr><td>Pcon-pSB1C3(X,S)</td><td>40</td><td>0.13</td><td>1.27</td></tr> |
</table> | </table> | ||
- | |||
- | |||
<h4>Electrophoresis</h4> | <h4>Electrophoresis</h4> | ||
- | <span class= | + | <span class="kyoto-author">Yasuda</span> |
<p>(1)</p> | <p>(1)</p> | ||
<table> | <table> | ||
Line 4,794: | Line 4,979: | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/c/c6/Kyoto-dms-0914_Elecrophoresis_No1.jpg"> | ||
<p>(2)</p> | <p>(2)</p> | ||
Line 4,816: | Line 5,002: | ||
<tr><td>17</td><td>9/12 DECARB-YCplac111(8)</td></tr> | <tr><td>17</td><td>9/12 DECARB-YCplac111(8)</td></tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/f/fc/Kyoto-dms-0914_Elecrophoresis_No2.jpg"> | ||
<p>(3)</p> | <p>(3)</p> | ||
Line 4,838: | Line 5,025: | ||
<tr><td>17</td><td>9/12 DiDECARB-YCplac111(4)</td></tr> | <tr><td>17</td><td>9/12 DiDECARB-YCplac111(4)</td></tr> | ||
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/6/6e/Kyoto-dms-0914_Elecrophoresis_No3.jpg"> | ||
<p>(4)</p> | <p>(4)</p> | ||
Line 4,860: | Line 5,048: | ||
<tr><td>17</td><td>9/12 DiDECARB-YCplac111(20)</td></tr> | <tr><td>17</td><td>9/12 DiDECARB-YCplac111(20)</td></tr> | ||
</table> | </table> | ||
- | + | <img src="https://static.igem.org/mediawiki/2014/9/9d/Kyoto-dms-0914_Elecrophoresis_No4.jpg"> | |
<h4>Miniprep</h4> | <h4>Miniprep</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
- | <tr><td>9/13 dddD- | + | <tr><td>9/13 dddD-pSB1C3(1)</td><td>645</td><td>1.82</td><td>1.70</td></tr> |
- | <tr><td>9/13 dddD- | + | <tr><td>9/13 dddD-pSB1C3(2)</td><td>660</td><td>1,69</td><td>1.74</td></tr> |
- | <tr><td>9/13 dddD- | + | <tr><td>9/13 dddD-pSB1C3(3)</td><td>635</td><td>1.87</td><td>1.57</td></tr> |
- | <tr><td>9/13 T7-RBS-His- | + | <tr><td>9/13 T7-RBS-His-pSB1C3(1)</td><td>440</td><td>1.65</td><td>1.23</td></tr> |
- | <tr><td>9/13 T7-RBS-His- | + | <tr><td>9/13 T7-RBS-His-pSB1C3(2)</td><td>450</td><td>1.92</td><td>1.46</td></tr> |
- | <tr><td>9/13 T7-RBS-His- | + | <tr><td>9/13 T7-RBS-His-pSB1C3(3)</td><td>190</td><td>1.44</td><td>1.26</td></tr> |
</table> | </table> | ||
Line 4,881: | Line 5,069: | ||
<h4>Restriction Enzyme Digestion</h4> | <h4>Restriction Enzyme Digestion</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | ||
- | <tr><td>9/14 dddD- | + | <tr><td>9/14 dddD-pSB1C3(1)</td><td>6</td><td>-</td><td>2</td><td>-</td><td>2</td><td>M</td><td>6</td><td>6</td><td>38</td><td>60</td></tr> |
- | <tr><td>9/14 dddD- | + | <tr><td>9/14 dddD-pSB1C3(2)</td><td>6</td><td>-</td><td>2</td><td>-</td><td>2</td><td>M</td><td>6</td><td>6</td><td>38</td><td>60</td></tr> |
- | <tr><td>9/14 dddD- | + | <tr><td>9/14 dddD-pSB1C3(3)</td><td>6</td><td>-</td><td>2</td><td>-</td><td>2</td><td>M</td><td>6</td><td>6</td><td>38</td><td>60</td></tr> |
- | <tr><td>9/14 T7-RBS-His- | + | <tr><td>9/14 T7-RBS-His-pSB1C3(1)</td><td>5</td><td>-</td><td>-</td><td>1</td><td>1</td><td>H</td><td>3</td><td>-</td><td>18</td><td>30</td></tr> |
- | <tr><td>9/14 T7-RBS-His- | + | <tr><td>9/14 T7-RBS-His-pSB1C3(2)</td><td>5</td><td>-</td><td>-</td><td>1</td><td>1</td><td>H</td><td>3</td><td>-</td><td>18</td><td>30</td></tr> |
- | <tr><td>9/14 T7-RBS-His- | + | <tr><td>9/14 T7-RBS-His-pSB1C3(3)</td><td>10</td><td>-</td><td>-</td><td>1</td><td>1</td><td>H</td><td>3</td><td>-</td><td>15</td><td>30</td></tr> |
</table> | </table> | ||
Line 4,903: | Line 5,091: | ||
<h4>Dephosphorylation</h4> | <h4>Dephosphorylation</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th><th>CIAP/(µL)</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th>CIAP/(µL)</th></tr> | ||
- | <tr><td>9/14 | + | <tr><td>9/14 pSB1C3(X,S)(gel extraction product)</td><td>20</td><td>0.25</td></tr> |
</table> | </table> | ||
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
- | <tr><td>9/14 | + | <tr><td>9/14 pSB1C3(X,S)(gel extraction product)</td><td>60</td><td>2.93</td><td>0.16</td></tr> |
</table> | </table> | ||
Line 4,917: | Line 5,105: | ||
<h4>Ligation</h4> | <h4>Ligation</h4> | ||
- | <span class= | + | <span class="kyoto-author">Yasuda</span> |
<table> | <table> | ||
<tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | <tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | ||
- | <tr><td>Pcon- | + | <tr><td>Pcon-pSB1C3(X,S)(Dephosphorylation product)</td><td>0.83</td><td>9/12 DECARB(X,S)(DNA purification product)</td><td>2.93</td><td>10x Lig Buffer</td><td>2</td><td>NEB T4 DNA Ligase</td><td>1</td><td>13.24</td><td>20</td></tr> |
- | <tr><td>Pcon- | + | <tr><td>Pcon-pSB1C3(X,S)(Dephosphorylation product)</td><td>0.83</td><td>9/12 DECARB(X,S)(DNA purification product)</td><td>3.66</td><td>10x Lig Buffer</td><td>2</td><td>NEB T4 DNA Ligase</td><td>1</td><td>12.51</td><td>20</td></tr> |
</table> | </table> | ||
Line 4,936: | Line 5,124: | ||
<h4>Colony PCR</h4> | <h4>Colony PCR</h4> | ||
- | <span class= | + | <span class="kyoto-author">Tatsui</span> |
<p>Master Mix(x24)</p> | <p>Master Mix(x24)</p> | ||
<table> | <table> | ||
<tr> | <tr> | ||
- | <th>Quick | + | <th>Quick Taq/(µL)</th> |
<th>VF/(µL)</th> | <th>VF/(µL)</th> | ||
<th>VR/(µL)</th> | <th>VR/(µL)</th> | ||
Line 4,983: | Line 5,171: | ||
<tr> | <tr> | ||
<td>1~8</td> | <td>1~8</td> | ||
- | <td>9/14 AT- | + | <td>9/14 AT-pSB1C3</td> |
</tr> | </tr> | ||
Line 4,990: | Line 5,178: | ||
<h4>Electrophoresis</h4> | <h4>Electrophoresis</h4> | ||
- | <span class= | + | <span class="kyoto-author">Tatsui</span> |
<table> | <table> | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
<tr><td>1</td><td>1kb ladder</td></tr> | <tr><td>1</td><td>1kb ladder</td></tr> | ||
- | <tr><td>2~9</td><td>9/14 AT- | + | <tr><td>2~9</td><td>9/14 AT-pSB1C3</td></tr> |
</table> | </table> | ||
Line 5,003: | Line 5,191: | ||
<h4>Gel Extraction</h4> | <h4>Gel Extraction</h4> | ||
- | <span class= | + | <span class="kyoto-author">Tatsui</span> |
<table> | <table> | ||
<tr><th>Lane</th><th>Sample</th></tr> | <tr><th>Lane</th><th>Sample</th></tr> | ||
<tr><td>1</td><td>1kb ladder</td></tr> | <tr><td>1</td><td>1kb ladder</td></tr> | ||
<tr><td>2</td><td>-</td></tr> | <tr><td>2</td><td>-</td></tr> | ||
- | <tr><td>3</td><td>9/14 dddD | + | <tr><td>3</td><td>9/14 dddD-pSB1C3(X,P)(After mutation)-(1)</td></tr> |
<tr><td>4</td><td>-</td></tr> | <tr><td>4</td><td>-</td></tr> | ||
<tr><td>5</td><td>1kb ladder</td></tr> | <tr><td>5</td><td>1kb ladder</td></tr> | ||
<tr><td>6</td><td>-</td></tr> | <tr><td>6</td><td>-</td></tr> | ||
- | <tr><td>7</td><td>9/14 dddD | + | <tr><td>7</td><td>9/14 dddD-pSB1C3(X,P)(After mutation)-(2)</td></tr> |
<tr><td>8</td><td>-</td></tr> | <tr><td>8</td><td>-</td></tr> | ||
<tr><td>9</td><td>1kb ladder</td></tr> | <tr><td>9</td><td>1kb ladder</td></tr> | ||
<tr><td>10</td><td>-</td></tr> | <tr><td>10</td><td>-</td></tr> | ||
- | <tr><td>11</td><td>9/14 dddD | + | <tr><td>11</td><td>9/14 dddD-pSB1C3(X,P)(After mutation)-(3)</td></tr> |
</table> | </table> | ||
Line 5,026: | Line 5,214: | ||
<h4>DNA Purification</h4> | <h4>DNA Purification</h4> | ||
- | <span class= | + | <span class="kyoto-author">Tatsui</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
- | <tr><td>9/14 dddD | + | <tr><td>9/14 dddD-pSB1C3(X,P)(After mutation)-(1)</td><td>1.7 (x50)</td><td>1.37</td><td>0.33</td></tr> |
- | <tr><td>9/14 dddD | + | <tr><td>9/14 dddD-pSB1C3(X,P)(After mutation)-(2)</td><td>1.6 (x50)</td><td>1.41</td><td>0.36</td></tr> |
- | <tr><td>9/14 dddD | + | <tr><td>9/14 dddD-pSB1C3(X,P)(After mutation)-(3)</td><td>2.2 (x50)</td><td>1.23</td><td>0.42</td></tr> |
- | <tr><td>T7-RBS- | + | <tr><td>T7-RBS-pSB1C3(S cut)</td><td>2.0 (x50)</td><td>2.11</td><td>0.34</td></tr> |
Line 5,043: | Line 5,231: | ||
- | <h4>Restriction Enzyme Digestion | + | <h4>Restriction Enzyme Digestion</h4> |
- | <span class= | + | <span class="kyoto-author">Murata</span> |
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | ||
- | <tr><td>9/13 T7-RBS- | + | <tr><td>9/13 T7-RBS-pSB1C3</td><td>7</td><td>-</td><td>-</td><td>1</td><td>1</td><td>H</td><td>3</td><td>-</td><td>9</td><td>30</td></tr> |
- | <tr><td>9/11 REDOX (DNA purification product)</td><td>10</td><td> | + | <tr><td>9/11 REDOX (DNA purification product)</td><td>10</td><td>1</td><td>-</td><td>-</td><td>1</td><td>H</td><td>3</td><td>-</td><td>15</td><td>30</td></tr> |
- | <tr><td>9/13 Pcon- | + | <tr><td>9/13 Pcon-pSB1C3</td><td>25</td><td>1</td><td>-</td><td>-</td><td>1</td><td>H</td><td>3</td><td>-</td><td>0</td><td>30</td></tr> |
- | <tr><td>9/15 T7-RBS- | + | <tr><td>9/15 T7-RBS-pSB1C3</td><td>25</td><td>-</td><td>-</td><td>-</td><td>1</td><td>M</td><td>3</td><td>-</td><td>1</td><td>30</td></tr> |
</table> | </table> | ||
+ | |||
+ | |||
Line 5,105: | Line 5,295: | ||
<h4>Gel Extraction</h4> | <h4>Gel Extraction</h4> | ||
- | <span class= | + | <span class="kyoto-author">Murata</span> |
<table> | <table> | ||
<tr><th>Lane</th><th>Sample</th></tr> | <tr><th>Lane</th><th>Sample</th></tr> | ||
<tr><td>1</td><td>1kb ladder</td></tr> | <tr><td>1</td><td>1kb ladder</td></tr> | ||
<tr><td>2</td><td>-</td></tr> | <tr><td>2</td><td>-</td></tr> | ||
- | <tr><td>3</td><td>9/14 T7-RBS-His- | + | <tr><td>3</td><td>9/14 T7-RBS-His-pSB1C3(S,P)-(1)</td></tr> |
<tr><td>4</td><td>-</td></tr> | <tr><td>4</td><td>-</td></tr> | ||
<tr><td>5</td><td>1kb ladder</td></tr> | <tr><td>5</td><td>1kb ladder</td></tr> | ||
<tr><td>6</td><td>-</td></tr> | <tr><td>6</td><td>-</td></tr> | ||
- | <tr><td>7</td><td>9/14 T7-RBS-His- | + | <tr><td>7</td><td>9/14 T7-RBS-His-pSB1C3(S,P)-(1)</td></tr> |
<tr><td>8</td><td>-</td></tr> | <tr><td>8</td><td>-</td></tr> | ||
<tr><td>9</td><td>1kb ladder</td></tr> | <tr><td>9</td><td>1kb ladder</td></tr> | ||
<tr><td>10</td><td>-</td></tr> | <tr><td>10</td><td>-</td></tr> | ||
- | <tr><td>11</td><td>9/14 T7-RBS-His- | + | <tr><td>11</td><td>9/14 T7-RBS-His-pSB1C3(S,P)-(1)</td></tr> |
</table> | </table> | ||
- | + | <img src="https://static.igem.org/mediawiki/2014/d/de/Kyoto-dms-0915_Gel_Extention_No1_before.jpg"> | |
+ | <img src="https://static.igem.org/mediawiki/2014/6/6e/Kyoto-dms-0915_Gel_Extention_No1_after.jpg"> | ||
<h4>DNA Purification</h4> | <h4>DNA Purification</h4> | ||
- | <span class= | + | <span class="kyoto-author">Yasuda</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
Line 5,133: | Line 5,324: | ||
<h4>Ligation</h4> | <h4>Ligation</h4> | ||
- | <span class= | + | <span class="kyoto-author"></span> |
<table> | <table> | ||
<tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | <tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | ||
Line 5,152: | Line 5,343: | ||
<h4>Liquid Culture</h4> | <h4>Liquid Culture</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Medium</th></tr> | <tr><th>Sample</th><th>Medium</th></tr> | ||
- | <tr><td>9/14 AT- | + | <tr><td>9/14 AT-pSB1C3 (Transformation product)-(1)</td><td>SOD</td></tr> |
- | <tr><td>9/14 AT- | + | <tr><td>9/14 AT-pSB1C3 (Transformation product)-(2)</td><td>SOD</td></tr> |
- | <tr><td>9/14 AT- | + | <tr><td>9/14 AT-pSB1C3 (Transformation product)-(3)</td><td>SOD</td></tr> |
</table> | </table> | ||
Line 5,165: | Line 5,356: | ||
<h4>Dephosphorylation</h4> | <h4>Dephosphorylation</h4> | ||
- | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th><th>CIAP/(µL)</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th>CIAP/(µL)</th></tr> | ||
- | <tr><td>9/15 T7-RBS- | + | <tr><td>9/15 T7-RBS-pSB1C3(P)(Restriction Enzyme Digestion product)</td><td>20</td><td>0.25</td></tr> |
- | <tr><td>9/13 | + | <tr><td>9/13 pSB1C3(E,P)(Gel extraction product)</td><td>20</td><td>0.25</td></tr> |
</table> | </table> | ||
Line 5,190: | Line 5,381: | ||
<h4>Gel Extraction</h4> | <h4>Gel Extraction</h4> | ||
- | <span class= | + | <span class="kyoto-author">Yasuda</span> |
<table> | <table> | ||
<tr><th>Lane</th><th>Sample</th></tr> | <tr><th>Lane</th><th>Sample</th></tr> | ||
<tr><td>1</td><td>1kb ladder</td></tr> | <tr><td>1</td><td>1kb ladder</td></tr> | ||
<tr><td>2</td><td>-</td></tr> | <tr><td>2</td><td>-</td></tr> | ||
- | <tr><td>3</td><td>9/14 T7-RBS-His- | + | <tr><td>3</td><td>9/14 T7-RBS-His-pSB1C3(S,P)-(1)</td></tr> |
<tr><td>4</td><td>-</td></tr> | <tr><td>4</td><td>-</td></tr> | ||
<tr><td>5</td><td>-</td></tr> | <tr><td>5</td><td>-</td></tr> | ||
<tr><td>6</td><td>-</td></tr> | <tr><td>6</td><td>-</td></tr> | ||
- | <tr><td>7</td><td>9/14 T7-RBS-His- | + | <tr><td>7</td><td>9/14 T7-RBS-His-pSB1C3(S,P)-(2)</td></tr> |
<tr><td>8</td><td>-</td></tr> | <tr><td>8</td><td>-</td></tr> | ||
<tr><td>9</td><td>-</td></tr> | <tr><td>9</td><td>-</td></tr> | ||
<tr><td>10</td><td>-</td></tr> | <tr><td>10</td><td>-</td></tr> | ||
- | <tr><td>11</td><td>9/14 T7-RBS-His- | + | <tr><td>11</td><td>9/14 T7-RBS-His-pSB1C3(S,P)-(3)</td></tr> |
</table> | </table> | ||
Line 5,209: | Line 5,400: | ||
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
- | <tr><td>9/14 T7-RBS-His- | + | <tr><td>9/14 T7-RBS-His-pSB1C3(S,P)-(1)</td><td>25</td><td>1.81</td><td>0.50</td></tr> |
- | <tr><td>9/14 T7-RBS-His- | + | <tr><td>9/14 T7-RBS-His-pSB1C3(S,P)-(2)</td><td>25</td><td>1.13</td><td>0.21</td></tr> |
- | <tr><td>9/14 T7-RBS-His- | + | <tr><td>9/14 T7-RBS-His-pSB1C3(S,P)-(3)</td><td>25</td><td>1.65</td><td>0.09</td></tr> |
</table> | </table> | ||
<h4>Restriction Enzyme Digestion</h4> | <h4>Restriction Enzyme Digestion</h4> | ||
- | <span class= | + | <span class="kyoto-author">Yasuda</span> |
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | ||
- | <tr><td>9/14 T7-RBS-His- | + | <tr><td>9/14 T7-RBS-His-pSB1C3 (Miniprep product)</td><td>13.63</td><td>-</td><td>-</td><td>3</td><td>3</td><td>H</td><td>9</td><td>-</td><td>61.37</td><td>90</td></tr> |
</table> | </table> | ||
Line 5,228: | Line 5,419: | ||
- | <h4>Transformation | + | <h4>Transformation</h4> |
- | <span class= | + | <span class="kyoto-author">Murata</span> |
+ | |||
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th><th colspan=2>CompetentCells/(µL)</th><th>Total/(µL)</th><th>Medium</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th colspan=2>CompetentCells/(µL)</th><th>Total/(µL)</th><th>Medium</th></tr> | ||
- | <tr><td> | + | <tr><td>AT-His-YCplac111</td><td>10</td><td>DH5α</td><td>2</td><td>12</td><td>CP</td></tr> |
+ | <tr><td>REDOX-YCplac111</td><td>10</td><td>DH5α</td><td>2</td><td>12</td><td>CP</td></tr> | ||
+ | <tr><td>DECARB-pSB1C3</td><td>10</td><td>DH5α</td><td>2</td><td>12</td><td>CP</td></tr> | ||
+ | <tr><td>DiDECARB-pSB1C3</td><td>10</td><td>DH5α</td><td>2</td><td>12</td><td>CP</td></tr> | ||
- | |||
- | |||
- | |||
- | |||
- | |||
</table> | </table> | ||
Line 5,245: | Line 5,435: | ||
<h4>Ligation</h4> | <h4>Ligation</h4> | ||
- | <span class= | + | <span class="kyoto-author"></span> |
<table> | <table> | ||
<tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | <tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | ||
- | <tr><td>9/15 T7-RBS(S,P)(Dephosphorylation product)</td><td>2.0</td><td>9/15 dddD( | + | <tr><td>9/15 T7-RBS(S,P)(Dephosphorylation product)</td><td>2.0</td><td>9/15 dddD(After mutation)(X,P)(Gel extraction product)</td><td>2.0</td><td>(x2)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>0</td><td>10</td></tr> |
- | <tr><td>9/15 | + | <tr><td>9/15 pSB1C3 (E,P)(Dephosphorylation product)</td><td>0.4</td><td>9/12 SAMmt (E,P)(DNA purification product)</td><td>2.9</td><td>(x2)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>0.7</td><td>10</td></tr> |
- | <tr><td>9/15 | + | <tr><td>9/15 pSB1C3 (E,P)(Dephosphorylation product)</td><td>1.3</td><td>9/15 REDOX (E,P)(DNA purification product)</td><td>2.4</td><td>(x2)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>0.3</td><td>10</td></tr> |
<tr><td>9/15 T7-RBS(S,P)(Gel extraction product)</td><td>2.6</td><td>9/15 AT His (X,P)(DNA purification product)</td><td>1.2</td><td>(x2)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>0.2</td><td>10</td></tr> | <tr><td>9/15 T7-RBS(S,P)(Gel extraction product)</td><td>2.6</td><td>9/15 AT His (X,P)(DNA purification product)</td><td>1.2</td><td>(x2)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>0.2</td><td>10</td></tr> | ||
Line 5,261: | Line 5,451: | ||
<h4>Transformation</h4> | <h4>Transformation</h4> | ||
- | <span class= | + | <span class="kyoto-author">Murata and Yasuda</span> |
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th><th colspan=2>CompetentCells/(µL)</th><th>Total/(µL)</th><th>Medium</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th colspan=2>CompetentCells/(µL)</th><th>Total/(µL)</th><th>Medium</th></tr> | ||
- | <tr><td>DECARB-YCplac111</td><td>2</td><td>DH5& | + | <tr><td>DECARB-YCplac111</td><td>2</td><td>DH5α</td><td>10</td><td>12</td><td>SOD</td></tr> |
- | <tr><td>DiDECARB-YCplac111</td><td>2</td><td>DH5& | + | <tr><td>DiDECARB-YCplac111</td><td>2</td><td>DH5α</td><td>10</td><td>12</td><td>SOD</td></tr> |
- | <tr><td>dddD( | + | <tr><td>dddD(After mutation)-T7-RBS-pSB1C3</td><td>2</td><td>DH5α</td><td>10</td><td>12</td><td>SOD</td></tr> |
- | <tr><td>SAMmt- | + | <tr><td>SAMmt-pSB1C3</td><td>2</td><td>DH5α</td><td>10</td><td>12</td><td>SOD</td></tr> |
- | <tr><td>REDOX- | + | <tr><td>REDOX-pSB1C3</td><td>2</td><td>DH5α</td><td>10</td><td>12</td><td>SOD</td></tr> |
- | <tr><td>AT-T7-RBS- | + | <tr><td>AT-T7-RBS-pSB1C3</td><td>2</td><td>DH5α</td><td>10</td><td>12</td><td>SOD</td></tr> |
</table> | </table> | ||
<h4>DNA Purification</h4> | <h4>DNA Purification</h4> | ||
- | <span class= | + | <span class="kyoto-author">Yasuda</span> |
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
Line 5,301: | Line 5,491: | ||
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
- | <tr><td>9/15 AT- | + | <tr><td>9/15 AT-pSB1C3-(1)</td><td>280</td><td>1.81</td><td>2.08</td></tr> |
- | <tr><td>9/15 AT- | + | <tr><td>9/15 AT-pSB1C3-(1)</td><td>205</td><td>1.64</td><td>1.32</td></tr> |
- | <tr><td>9/15 AT- | + | <tr><td>9/15 AT-pSB1C3-(1)</td><td>320</td><td>1.66</td><td>1.61</td></tr> |
</table> | </table> | ||
Line 5,310: | Line 5,500: | ||
<span class="kyoto-author">Tatsui and Nakamura</span> | <span class="kyoto-author">Tatsui and Nakamura</span> | ||
- | <p | + | <p>9/14 dddD-pSB1C3(Miniprep product)-(1)<br> |
- | + | (1)dddD sequence VF2</p> | |
<table> | <table> | ||
<tr> | <tr> | ||
Line 5,332: | Line 5,522: | ||
- | <p | + | <p>(2)dddD sequence No.1</p> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 5,352: | Line 5,542: | ||
</table> | </table> | ||
- | <p | + | <p>(3)dddD sequence No.2</p> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 5,373: | Line 5,563: | ||
- | <p | + | <p>(4)dddD sequence No.3</p> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 5,394: | Line 5,584: | ||
- | <p | + | <p>(5)dddD sequence No.4</p> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 5,415: | Line 5,605: | ||
- | <p | + | <p>(6)dddD sequence No.5</p> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 5,436: | Line 5,626: | ||
- | <p | + | <p>(7)dddD sequence No.6</p> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 5,463: | Line 5,653: | ||
<th>Annealing</th> | <th>Annealing</th> | ||
<th>Extension</th> | <th>Extension</th> | ||
- | <th>Final | + | <th>Final Extension</th> |
<th>Cooling</th> | <th>Cooling</th> | ||
</tr> | </tr> | ||
Line 5,480: | Line 5,670: | ||
<td>4min</td> | <td>4min</td> | ||
<td>1min</td> | <td>1min</td> | ||
- | <td> | + | <td>Infinity</td> |
</tr> | </tr> | ||
</table> | </table> | ||
Line 5,490: | Line 5,680: | ||
- | <p | + | <p>9/14 dddD-pSB1C3(Miniprep product)-(2)</p> |
- | <p | + | <p>(1)dddD sequence VF2</p> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 5,512: | Line 5,702: | ||
- | <p | + | <p>(2)dddD sequence No.1</p> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 5,532: | Line 5,722: | ||
</table> | </table> | ||
- | <p | + | <p>(3)dddD sequence No.2</p> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 5,553: | Line 5,743: | ||
- | <p | + | <p>(4)dddD sequence No.3</p> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 5,574: | Line 5,764: | ||
- | <p | + | <p>(5)dddD sequence No.4</p> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 5,595: | Line 5,785: | ||
- | <p | + | <p>(6)dddD sequence No.5</p> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 5,616: | Line 5,806: | ||
- | <p | + | <p>(7)dddD sequence No.6</p> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 5,637: | Line 5,827: | ||
- | <p | + | <p>9/15 AT-pSB1C3(Miniprep product)-(1)</p> |
- | <p | + | <p>(1)VF</p> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 5,658: | Line 5,848: | ||
</table> | </table> | ||
- | <p | + | <p>(2)AT for Sequence</p> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 5,684: | Line 5,874: | ||
<th>Annealing</th> | <th>Annealing</th> | ||
<th>Extension</th> | <th>Extension</th> | ||
- | <th>Final | + | <th>Final Extension</th> |
<th>Cooling</th> | <th>Cooling</th> | ||
</tr> | </tr> | ||
Line 5,701: | Line 5,891: | ||
<td>4min</td> | <td>4min</td> | ||
<td>1min</td> | <td>1min</td> | ||
- | <td> | + | <td>Infinity</td> |
</tr> | </tr> | ||
</table> | </table> | ||
Line 5,708: | Line 5,898: | ||
- | <h4>Dephosphorylation | + | <h4>Dephosphorylation</h4> |
<span class="kyoto-author">Honda</span> | <span class="kyoto-author">Honda</span> | ||
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th></tr> | <tr><th colspan=2>Sample/(µL)</th></tr> | ||
- | <tr><td>1.</td><td> | + | <tr><td>1.*(written in magnetosome)</td><td>*</td></tr> |
- | <tr><td>2.</td><td> | + | <tr><td>2.*</td><td>*</td></tr> |
- | <tr><td>3.9/15 T7-RBS- | + | <tr><td>3.9/15 T7-RBS-pSB1C3(P)</td><td>30</td></tr> |
- | <tr><td>4.9/15 T7-RBS- | + | <tr><td>4.9/15 T7-RBS-pSB1C3(S,P)</td><td>30</td></tr> |
- | <tr><td>5.9/15 T7-RBS- | + | <tr><td>5.9/15 T7-RBS-pSB1C3(S,P)</td><td>30</td></tr> |
- | <tr><td>6.9/15 T7-RBS- | + | <tr><td>6.9/15 T7-RBS-pSB1C3(S,P)</td><td>30</td></tr> |
- | <tr><td>7.9/15 T7-RBS- | + | <tr><td>7.9/15 T7-RBS-pSB1C3(S,P)</td><td>30</td></tr> |
</table> | </table> | ||
Line 5,728: | Line 5,918: | ||
<table> | <table> | ||
<tr> | <tr> | ||
- | <th>Quick | + | <th>Quick Taq/(µL)</th> |
<th>M13 Ew/(µL)</th> | <th>M13 Ew/(µL)</th> | ||
<th>M13 Rv/(µL)</th> | <th>M13 Rv/(µL)</th> | ||
Line 5,742: | Line 5,932: | ||
</tr> | </tr> | ||
</table> | </table> | ||
- | + | <img src="https://static.igem.org/mediawiki/2014/d/d7/Kyoto-dms-0916_Colony_PCR_No1.jpg"> | |
+ | <img src="https://static.igem.org/mediawiki/2014/0/00/Kyoto-dms-0916_Colony_PCR_No2.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/9/95/Kyoto-dms-0916_Colony_PCR_No3.jpg"> | ||
<h4>Electrophoresis</h4> | <h4>Electrophoresis</h4> | ||
<span class="kyoto-author">Yasuda</span> | <span class="kyoto-author">Yasuda</span> | ||
- | <p | + | <p>(1)</p> |
<table> | <table> | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
Line 5,754: | Line 5,946: | ||
</table> | </table> | ||
- | <p | + | <p>(2)</p> |
<table> | <table> | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
Line 5,762: | Line 5,954: | ||
</table> | </table> | ||
- | <p | + | <p>(3)</p> |
<table> | <table> | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
Line 5,769: | Line 5,961: | ||
</table> | </table> | ||
- | <p | + | <p>(4)</p> |
<table> | <table> | ||
<tr><th>Lane</th><th colspan=3>Sample</th></tr> | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
Line 5,784: | Line 5,976: | ||
<h4>Gel Extraction</h4> | <h4>Gel Extraction</h4> | ||
<span class="kyoto-author">Yamauchi</span> | <span class="kyoto-author">Yamauchi</span> | ||
- | <p | + | <p>(1)</p> |
<table> | <table> | ||
<tr><th>Lane</th><th>Sample</th></tr> | <tr><th>Lane</th><th>Sample</th></tr> | ||
Line 5,797: | Line 5,989: | ||
<tr><td>9</td><td>1kb ladder</td></tr> | <tr><td>9</td><td>1kb ladder</td></tr> | ||
<tr><td>10</td><td>-</td></tr> | <tr><td>10</td><td>-</td></tr> | ||
- | <tr><td>11</td><td>9/15 | + | <tr><td>11</td><td>9/15 pSB1C3(E,P)</td></tr> |
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/9/92/Kyoto-dms-0916_Gel_Extention_No1_before.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/f/fa/Kyoto-dms-0916_Gel_Extention_No1_after.jpg"> | ||
- | <p | + | <p>(2)</p> |
<table> | <table> | ||
<tr><th>Lane</th><th>Sample</th></tr> | <tr><th>Lane</th><th>Sample</th></tr> | ||
<tr><td>1</td><td>1kb ladder</td></tr> | <tr><td>1</td><td>1kb ladder</td></tr> | ||
<tr><td>2</td><td>-</td></tr> | <tr><td>2</td><td>-</td></tr> | ||
- | <tr><td>3</td><td>9/15 T7-RBS- | + | <tr><td>3</td><td>9/15 T7-RBS-pSB1C3(P)</td></tr> |
<tr><td>4</td><td>-</td></tr> | <tr><td>4</td><td>-</td></tr> | ||
<tr><td>5</td><td>1kb ladder</td></tr> | <tr><td>5</td><td>1kb ladder</td></tr> | ||
<tr><td>6</td><td>-</td></tr> | <tr><td>6</td><td>-</td></tr> | ||
- | <tr><td>7</td><td>9/15 T7-RBS- | + | <tr><td>7</td><td>9/15 T7-RBS-pSB1C3(S,P)</td></tr> |
<tr><td>8</td><td>-</td></tr> | <tr><td>8</td><td>-</td></tr> | ||
<tr><td>9</td><td>1kb ladder</td></tr> | <tr><td>9</td><td>1kb ladder</td></tr> | ||
<tr><td>10</td><td>-</td></tr> | <tr><td>10</td><td>-</td></tr> | ||
- | <tr><td>11</td><td>9/15 T7-RBS- | + | <tr><td>11</td><td>9/15 T7-RBS-pSB1C3(S,P)</td></tr> |
</table> | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/3/32/Kyoto-dms-0916_Gel_Extention_No2_before.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/c/c0/Kyoto-dms-0916_Gel_Extention_No2_after.jpg"> | ||
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
- | <tr><td>7. 9/15 T7-RBS- | + | <tr><td>7. 9/15 T7-RBS-pSB1C3(S,P)</td><td>90</td><td>1.40</td><td>0.44</td></tr> |
</table> | </table> | ||
- | <p | + | <p>(3)</p> |
<table> | <table> | ||
<tr><th>Lane</th><th>Sample</th></tr> | <tr><th>Lane</th><th>Sample</th></tr> | ||
Line 5,829: | Line 6,025: | ||
<tr><td>2</td><td>1kb ladder</td></tr> | <tr><td>2</td><td>1kb ladder</td></tr> | ||
<tr><td>3</td><td>-</td></tr> | <tr><td>3</td><td>-</td></tr> | ||
- | <tr><td>4</td><td>9/15 T7-RBS- | + | <tr><td>4</td><td>9/15 T7-RBS-pSB1C3(S,P)</td></tr> |
<tr><td>5</td><td>-</td></tr> | <tr><td>5</td><td>-</td></tr> | ||
<tr><td>6</td><td>1kb ladder</td></tr> | <tr><td>6</td><td>1kb ladder</td></tr> | ||
</table> | </table> | ||
- | + | <img src="https://static.igem.org/mediawiki/2014/0/07/Kyoto-dms-0916_Gel_Extention_No3_before.jpg"> | |
+ | <img src="https://static.igem.org/mediawiki/2014/0/09/Kyoto-dms-0916_Gel_Extention_No3_after.jpg"> | ||
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
- | <tr><td>4. 9/15 T7-RBS- | + | <tr><td>4. 9/15 T7-RBS-pSB1C3(S,P)</td><td>80</td><td>1.30</td><td>1.02</td></tr> |
</table> | </table> | ||
Line 5,846: | Line 6,043: | ||
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
- | <tr><td>9/15 T7-RBS- | + | <tr><td>9/15 T7-RBS-pSB1C3(S,P)(Dephosphorylation product)</td><td>0.7(x50)</td><td>1.81</td><td>1.13</td></tr> |
- | <tr><td>9/15 | + | <tr><td>9/15 pSB1C3(E,P)(Dephosphorylation product)</td><td>1.1(x50)</td><td>1.59</td><td>1.25</td></tr> |
</table> | </table> | ||
Line 5,858: | Line 6,055: | ||
<tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | <tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | ||
- | <tr><td>9/16 T7-RBS(S,P)(DNA purification product)</td><td>1.7</td><td>9/15 dddD( | + | <tr><td>9/16 T7-RBS(S,P)(DNA purification product)</td><td>1.7</td><td>9/15 dddD(After mutation)(X,P)(gel extraction product)</td><td>2</td><td>(x2)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>0.3</td><td>10</td></tr> |
- | <tr><td>9/16 | + | <tr><td>9/16 pSB1C3 (E,P)(DNA purification product)</td><td>0.55</td><td>9/15 SAMmt (E,P)(gel extraction product)</td><td>2.9</td><td>(x2)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>0.55</td><td>10</td></tr> |
- | <tr><td>9/16 | + | <tr><td>9/16 pSB1C3 (E,P)(DNA purification product)</td><td>1.6</td><td>9/15 REDOX (E,P)(gel extraction product)</td><td>2.2</td><td>(x2)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>0.2</td><td>10</td></tr> |
<tr><td>9/16 T7-RBS-His(S,P)(DNA purification product)</td><td>2.6</td><td>9/15 AT His(X,P)(gel extraction product)</td><td>1.2</td><td>(x2)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>0.2</td><td>10</td></tr> | <tr><td>9/16 T7-RBS-His(S,P)(DNA purification product)</td><td>2.6</td><td>9/15 AT His(X,P)(gel extraction product)</td><td>1.2</td><td>(x2)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>0.2</td><td>10</td></tr> | ||
Line 5,873: | Line 6,070: | ||
<table> | <table> | ||
<tr><th colspan=2>Sample/(µL)</th><th>CIAP</th><th>Total</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th>CIAP</th><th>Total</th></tr> | ||
- | <tr><td>9/13 | + | <tr><td>9/13 pSB1C3 (X,S)(gel extraction product)</td><td>16</td><td>0.2</td><td>16.2</td></tr> |
</table> | </table> | ||
Line 5,884: | Line 6,081: | ||
<tr><td>9/16 AT-His-T7-RBS-His</td><td>-</td><td>DH5α</td><td>-</td><td>-</td><td>-</td></tr> | <tr><td>9/16 AT-His-T7-RBS-His</td><td>-</td><td>DH5α</td><td>-</td><td>-</td><td>-</td></tr> | ||
- | <tr><td>9/16 REDOX- | + | <tr><td>9/16 REDOX-pSB1C3</td><td>-</td><td>DH5α</td><td>-</td><td>-</td><td>-</td></tr> |
- | <tr><td>9/16 SAMmt- | + | <tr><td>9/16 SAMmt-pSB1C3</td><td>-</td><td>DH5α</td><td>-</td><td>-</td><td>-</td></tr> |
- | <tr><td>9/16 dddD | + | <tr><td>9/16 dddD-T7-RBS(After mutation)</td><td>-</td><td>DH5α</td><td>-</td><td>-</td><td>-</td></tr> |
<tr><td>9/16 AT-His-YCplac111</td><td>-</td><td>DH5α</td><td>-</td><td>-</td><td>-</td></tr> | <tr><td>9/16 AT-His-YCplac111</td><td>-</td><td>DH5α</td><td>-</td><td>-</td><td>-</td></tr> | ||
Line 5,904: | Line 6,101: | ||
<table> | <table> | ||
<tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
- | <tr><td>9/16 | + | <tr><td>9/16 pSB1C3(X,S)(Dephosphorylation product)</td><td>35</td><td>1.69</td><td>0.56</td></tr> |
</table> | </table> | ||
Line 5,921: | Line 6,118: | ||
<h4>PCR</h4> | <h4>PCR</h4> | ||
<span class="kyoto-author">Yasuda</span> | <span class="kyoto-author">Yasuda</span> | ||
- | <p | + | <p>(1)</p> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 5,993: | Line 6,190: | ||
<tr><td>9/11 OE-PCR SAMmt 2nd-(2)</td><td>8.2</td><td>0.5</td><td>-</td><td>-</td><td>0.5</td><td>H</td><td>1.5</td><td>0</td><td>4.3</td><td>15</td></tr> | <tr><td>9/11 OE-PCR SAMmt 2nd-(2)</td><td>8.2</td><td>0.5</td><td>-</td><td>-</td><td>0.5</td><td>H</td><td>1.5</td><td>0</td><td>4.3</td><td>15</td></tr> | ||
- | <tr><td>9/15 AT- | + | <tr><td>9/15 AT-pSB1C3-(3)(Miniprep product)</td><td>9.8</td><td>-</td><td>1</td><td>-</td><td>1</td><td>M</td><td>3</td><td>3</td><td>12.2</td><td>30</td></tr> |
<tr><td>Control</td><td>0.5</td><td>-</td><td>-</td><td>-</td><td>-</td><td>M</td><td>1</td><td>1</td><td>7.5</td><td>10</td></tr> | <tr><td>Control</td><td>0.5</td><td>-</td><td>-</td><td>-</td><td>-</td><td>M</td><td>1</td><td>1</td><td>7.5</td><td>10</td></tr> | ||
Line 6,004: | Line 6,201: | ||
<tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | <tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | ||
- | <tr><td> | + | <tr><td>pSB1C3</td><td>1.1</td><td>9/12 DECARB(X,E)(DNA purification product)</td><td>2.9</td><td>(x2)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>0</td><td>10</td></tr> |
- | <tr><td> | + | <tr><td>pSB1C3</td><td>0.85</td><td>9/12 DiDECARB(X,E)(DNA purification product)</td><td>2.2</td><td>(x2)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>0.95</td><td>10</td></tr> |
</table> | </table> | ||
Line 6,016: | Line 6,213: | ||
<tr><th colspan=2>Sample/(µL)</th><th colspan=2>CompetentCells/(µL)</th><th>Total/(µL)</th><th>Medium</th></tr> | <tr><th colspan=2>Sample/(µL)</th><th colspan=2>CompetentCells/(µL)</th><th>Total/(µL)</th><th>Medium</th></tr> | ||
- | <tr><td>9/16 DECARB- | + | <tr><td>9/16 DECARB-pSB1C3</td><td>1</td><td>DH5α</td><td>10</td><td>11</td><td>-</td></tr> |
- | <tr><td>9/16 DiDECARB- | + | <tr><td>9/16 DiDECARB-pSB1C3</td><td>1</td><td>DH5α</td><td>10</td><td>11</td><td>-</td></tr> |
</table> | </table> | ||
Line 6,032: | Line 6,229: | ||
<h3>9/17</h3> | <h3>9/17</h3> | ||
+ | <h4>Cycle PCR</h4> | ||
+ | <span class="kyoto-author">Murata and Nakamura</span> | ||
+ | <p>16 Cycle</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>BigDye Terminator v3.1 Cycle Sequencing RR-24/(µL)</th> | ||
+ | <th>BigDye Terminator v3.1 v1.1 5x Sequencing Buffer/(µL)</th> | ||
+ | <th>Primer #1/(pmol)</th> | ||
+ | <th>Templete DNA #2/(ng)</th> | ||
+ | <th>MilliQ /(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>1</td> | ||
+ | <td>1.6</td> | ||
+ | <td>75-150</td> | ||
+ | <td>up to 10</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <P> | ||
+ | <br>(1) | ||
+ | <br> | ||
+ | #1: VF2<br> | ||
+ | #2: 9/11 dddD (miniprep product) 1µL<br> | ||
+ | <br> | ||
+ | #1: Sequence No.5<br> | ||
+ | #2: 9/11 dddD (miniprep product) 1µL<br> | ||
+ | <br> | ||
+ | <br>(2) | ||
+ | <br> | ||
+ | #1: VF2<br> | ||
+ | #2: 9/14 dddD(After mutation)(miniprep product) 0.25µL<br> | ||
+ | <br> | ||
+ | #1: VR<br> | ||
+ | #2: 9/14 dddD(After mutation)(miniprep product) 0.25µL<br> | ||
+ | <br> | ||
+ | <br>(3) | ||
+ | <br> | ||
+ | #1: VF2<br> | ||
+ | #2: 9/14 T7-RBS-His-pSB1C3 (miniprep product) 0.34µL<br> | ||
+ | <br> | ||
+ | #1: Sequence No.5<br> | ||
+ | #2: 9/14 T7-RBS-His-pSB1C3 (miniprep product) 0.34µL<br> | ||
+ | <br> | ||
+ | <br>(4) | ||
+ | <br> | ||
+ | #1: VR<br> | ||
+ | #2: 9/16 AT-pSB1C3-(1) (miniprep product) 0.5µL<br> | ||
+ | <br> | ||
+ | #1: VF2<br> | ||
+ | #2: 9/16 AT-pSB1C3-(1) (miniprep product) 0.5µL<br> | ||
+ | <br> | ||
+ | #1: Sequence No.1<br> | ||
+ | #2: 9/16 AT-pSB1C3-(1) (miniprep product) 0.5µL<br> | ||
+ | <br> | ||
+ | #1: Secuence No.2<br> | ||
+ | #2: 9/16 AT-pSB1C3-(1) (miniprep product) 0.5µL<br> | ||
+ | |||
+ | </P> | ||
+ | |||
+ | |||
+ | |||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Final Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>96°C</td> | ||
+ | <td>96°C</td> | ||
+ | <td>50°C</td> | ||
+ | <td>60°C</td> | ||
+ | <td>4°C</td> | ||
+ | <td>4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>5sec</td> | ||
+ | <td>4min</td> | ||
+ | <td>1min</td> | ||
+ | <td>Infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <h4>Gel Extraction</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | <table> | ||
+ | <tr><th>Lane</th><th>Sample</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td></tr> | ||
+ | <tr><td>2</td><td>Control</td></tr> | ||
+ | <tr><td>3</td><td>-</td></tr> | ||
+ | <tr><td>4</td><td>9/16 AT-pSB1C3(X,P)</td></tr> | ||
+ | <tr><td>5</td><td>-</td></tr> | ||
+ | <tr><td>6</td><td>-</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/d/d7/Kyoto-dms-0917_Gel_Extention_No1_before.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/e/e5/Kyoto-dms-0917_Gel_Extention_No1_after.jpg"> | ||
+ | |||
+ | <h4>DNA Purification</h4> | ||
+ | <span class="kyoto-author">Murata and Yasuda</span> | ||
+ | <table> | ||
+ | <tr><th>Sample</th><th>Concentration/(µg/mL)</th></tr> | ||
+ | <tr><td>9/12 SAMmt (E,P)</td><td>75</td></tr> | ||
+ | <tr><td>9/16 SAMmt (PCR product)</td><td>140</td></tr> | ||
+ | <tr><td>9/16 DECARB (PCR product)</td><td>230</td></tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | <h4>PCR</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>10x Buffer for KOD-Plus-Ver.2</th> | ||
+ | <th>2mM dNTPs</th> | ||
+ | <th>25mM MgSO4</th> | ||
+ | <th>Primer(10µM each) #1</th> | ||
+ | <th>Template DNA #2</th> | ||
+ | <th>KOD-Plus-(1U/µL)</th> | ||
+ | <th>MilliQ</th> | ||
+ | <th>Total</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5µL</td> | ||
+ | <td>5µL</td> | ||
+ | <td>3µL</td> | ||
+ | <td>2µL</td> | ||
+ | <td>0.5µL</td> | ||
+ | <td>1µL</td> | ||
+ | <td>31.5µL</td> | ||
+ | <td>50µL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <p> | ||
+ | <br>(1)<br> | ||
+ | #1: Pre-REDOX, Suf-REDOX<br> | ||
+ | #2: 9/11 REDOX (DNA purification product)<br> | ||
+ | |||
+ | <br>(2)<br> | ||
+ | #1: Pre-REDOX, Rv-REDOX cloning for His<br> | ||
+ | #2: 9/11 REDOX (DNA purification product)<br> | ||
+ | |||
+ | <br>(3)<br> | ||
+ | #1: DiDECARB OE-A, DiDECARB OE-D<br> | ||
+ | #2: 9/11 DiDECARB (for OE-PCR 2nd)(9/12 DNA purification product)<br> | ||
+ | |||
+ | |||
+ | </p> | ||
+ | |||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Final Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>98°C</td> | ||
+ | <td>98°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>72°C</td> | ||
+ | <td>72°C</td> | ||
+ | <td>12°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>10sec</td> | ||
+ | <td>1min</td> | ||
+ | <td>5min</td> | ||
+ | <td>Infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 25cycle</p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <h4>DNA Purification</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr><th>Sample</th><th>Concentration/(µg/mL)</th></tr> | ||
+ | |||
+ | <tr><td>9/17 REDOX PCR</td><td>435</td></tr> | ||
+ | |||
+ | <tr><td>9/17 REDOX-His PCR</td><td>365</td></tr> | ||
+ | |||
+ | <tr><td>9/17 DiDECARB PCR</td><td>230</td</tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | |||
+ | <h4>Colony PCR</h4> | ||
+ | <span class="kyoto-author">Honda,Nakamura and Matsumoto</span> | ||
+ | <p>Master Mix</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Quick Taq/(µL)</th> | ||
+ | <th>VF2/(µL)</th> | ||
+ | <th>VR/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>85</td> | ||
+ | <td>3.4</td> | ||
+ | <td>3.4</td> | ||
+ | <td>78.2</td> | ||
+ | <td>170</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>94°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>68°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>30sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>2min30sec</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Number</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1-2</td> | ||
+ | <td>9/15 AT-His-T7-RBS-His (1)-(2)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3-7</td> | ||
+ | <td>9/16 AT-His-T7-RBS-His (1)-(5)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>8-9</td> | ||
+ | <td>9/16 dddD-T7-RBS (After mutation)(1)-(2)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10-16</td> | ||
+ | <td>9/16 SAMmt-pSB1C3 (1)-(7)</td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | |||
+ | |||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | <table> | ||
+ | <tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | ||
+ | |||
+ | <tr><td>9/17 REDOX-His (DNA purification product)</td><td>5.5</td><td>-</td><td>1</td><td>-</td><td>1</td><td>M</td><td>3</td><td>3</td><td>16.5</td><td>30</td></tr> | ||
+ | |||
+ | <tr><td>9/17 REDOX (DNA purification product)</td><td>4.6</td><td>-</td><td>1</td><td>-</td><td>1</td><td>M</td><td>3</td><td>3</td><td>17.4</td><td>30</td></tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | |||
+ | <h4>Ligation</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th colspan=2>Buffer/(µL)</th><th colspan=2>Ligase/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</tr> | ||
+ | |||
+ | <tr><td>9/16 T7-RBS(S,P) (DNA purification product)</td><td>1.4</td><td>9/17 AT(X,P) (DNA purification product)</td><td>2.3</td><td>(x2)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>0.3</td><td>10</td></tr> | ||
+ | |||
+ | <tr><td>YCplac111(SmaI cut)</td><td>1</td><td>9/17 REDOX-His (DNA purification product)</td><td>0.6</td><td>(x2)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>2.39</td><td>10</td></tr> | ||
+ | |||
+ | <tr><td>9/16 pSB1C3(E,P) (DNA purification product)</td><td>0.8</td><td>9/17 SAMmt(E,P) (DNA purification product)</td><td>1.9</td><td>(x2)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>0.8</td><td>10</td></tr> | ||
+ | |||
+ | <tr><td>YCplac111(SmaI cut)</td><td>1</td><td>-</td><td>-</td><td>(x2)Buffer</td><td>5</td><td>T4 Ligase</td><td>1</td><td>3</td><td>10</td></tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <h4>Transformation</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr><th colspan=2>Sample/(µL)</th><th colspan=2>CompetentCells/(µL)</th><th>Total/(µL)</th><th>Medium</th></tr> | ||
+ | |||
+ | <tr><td>9/17 AT-T7-RBS (Ligation product)</td><td>2</td><td>DH5α</td><td>10</td><td>12</td><td>SOC</td></tr> | ||
+ | <tr><td>9/17 REDOX-His-YCplac111 (Ligation product)</td><td>2</td><td>DH5α</td><td>10</td><td>12</td><td>SOC</td></tr> | ||
+ | <tr><td>9/17 SAMmt-pSB1C3 (Ligation product)</td><td>2</td><td>DH5α</td><td>10</td><td>12</td><td>SOC</td></tr> | ||
+ | <tr><td>9/17 Control (SmaX3, Ligation product)</td><td>2</td><td>DH5α</td><td>10</td><td>12</td><td>SOC</td></tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | <h4>Make Control (for 9/18 UV-Spectrum)</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | <br> | ||
+ | <p>Add MTOB (1.6mg) to ethanenitrile (313.4µL)</p> | ||
+ | |||
+ | <h4>Electrophoresis</h4> | ||
+ | <span class="kyoto-author">Yasuda and Honda</span> | ||
+ | <p>(1)</p> | ||
+ | <table> | ||
+ | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
+ | <tr><td>1-2</td><td>9/15 AT-His-T7-RBS His (1)(2)</td></tr> | ||
+ | <tr><td>3-7</td><td>9/15 AT-His-T7-RBS His (1)-(5)</td></tr> | ||
+ | <tr><td>8</td><td>9/16 dddD-T7-RBS(After mutation)(1)</td></tr> | ||
+ | <tr><td>9</td><td>1kb ladder</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/a/a8/Kyoto-dms-0917_Elecrophoresis_No1.jpg"> | ||
+ | |||
+ | <p>(2)</p> | ||
+ | <table> | ||
+ | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td></tr> | ||
+ | <tr><td>2</td><td>9/16 dddD-T7-RBS(After mutation)(2)</td></tr> | ||
+ | <tr><td>3-8</td><td>9/16 SAMmt-pSB1C3 (1)-(6)</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/9/9c/Kyoto-dms-0917_Elecrophoresis_No2.jpg"> | ||
+ | |||
+ | <p>(3)</p> | ||
+ | <table> | ||
+ | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td></tr> | ||
+ | <tr><td>2</td><td>9/16 SAMmt-pSB1C3 (7)</td></tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | |||
+ | <h4>Liquid Culture</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr><th>Sample</th></tr> | ||
+ | <tr><td>9/16 AT His-T7-RBS His (2)-(4)</td></tr> | ||
+ | <tr><td>9/16 SAMmt-pSB1C3 (2)(4)</td></tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | <h4>DNA Purification</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | <table> | ||
+ | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>9/17 REDOX His (X,P)</td><td>1.6</td><td>1.74</td><td>-</td></tr> | ||
+ | <tr><td>9/17 REDOX (X,P)(PCR product)</td><td>1.3</td><td>2.43</td><td>-</td></tr> | ||
+ | </table> | ||
Line 6,038: | Line 6,600: | ||
</div> | </div> | ||
+ | <div> | ||
+ | |||
+ | <a name="0918" class="kyoto-jump"></a> | ||
+ | <h3>9/18</h3> | ||
+ | |||
+ | |||
+ | <h4>Miniprep</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | <table> | ||
+ | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>9/15 T7-AT-His (1)</td><td>200</td><td>1.54</td><td>1.60</td></tr> | ||
+ | <tr><td>9/15 T7-AT-His (2)</td><td>180</td><td>1.64</td><td>2.02</td></tr> | ||
+ | <tr><td>9/16 T7-AT-His (2)</td><td>140</td><td>1.84</td><td>2.09</td></tr> | ||
+ | <tr><td>9/16 T7-AT-His (3)</td><td>140</td><td>1.71</td><td>1.82</td></tr> | ||
+ | <tr><td>9/16 T7-AT-His (4)</td><td>145</td><td>1.83</td><td>2.16</td></tr> | ||
+ | <tr><td>9/16 SAMmt-pSB1C3 (2)</td><td>225</td><td>1.64</td><td>1.71</td></tr> | ||
+ | <tr><td>9/16 SAMmt-pSB1C3 (4)</td><td>190</td><td>1.75</td><td>2.12</td></tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <h4>Colony PCR</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <p>Master Mix</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Quick Taq/(µL)</th> | ||
+ | <th>Fw M13/(µL)</th> | ||
+ | <th>Rv M13/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>45</td> | ||
+ | <td>1.8</td> | ||
+ | <td>1.8</td> | ||
+ | <td>41.4</td> | ||
+ | <td>90</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>94°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>68°C</td> | ||
+ | <td>12°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>30sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>90sec</td> | ||
+ | <td>Infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | |||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Number</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>9/16 REDOX-(1)-1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>9/16 DiDECARBO-(1)-1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>9/16 DiDECARBO-(1)-2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>9/16 DiDECARBO-(1)-3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>9/16 DiDECARBO-(1)-4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td>9/17 AT-T7-RBS-1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>7</td> | ||
+ | <td>9/17 REDOX-His-YCplac111-1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>8</td> | ||
+ | <td>9/17 REDOX-His-YCplac111-2</td> | ||
+ | </tr><tr> | ||
+ | <td>9</td> | ||
+ | <td>9/17 REDOX-His-YCplac111-3</td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | |||
+ | <table> | ||
+ | <tr><th>Lane</th><th>Sample</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td></tr> | ||
+ | <tr><td>2</td><td>9/16 REDOX-YCplac111 (1)</td></tr> | ||
+ | <tr><td>3-6</td><td>9/16 DiDECARB-YCplac111 (1)-(4)</td></tr> | ||
+ | </table> | ||
+ | |||
+ | <table> | ||
+ | <tr><th>Lane</th><th>Sample</th></tr> | ||
+ | <tr><td>1</td><td>-</td></tr> | ||
+ | <tr><td>2</td><td>1kb ladder</td></tr> | ||
+ | <tr><td>3</td><td>9/17 AT-T7-RBS (1)</td></tr> | ||
+ | <tr><td>4-6</td><td>REDOX-His=YCplac111 (1)-(3)</td></tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2014/2/2d/Kyoto-dms-0917_Colony_PCR_No1.jpg"> | ||
+ | |||
+ | |||
+ | <h4>Liquid Culture</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th><th>Medium</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/17 REDOX-His-YCplac111-(1)</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/17 REDOX-His-YCplac111-(2)</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/14 AT-pSB1C3-(1)</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | |||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(µL)</th> | ||
+ | <th>EcoR1/(µL)</th> | ||
+ | <th>Xba1/(µL)</th> | ||
+ | <th>Spe1/(µL)</th> | ||
+ | <th>Pst1/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th>BSA/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/15 AT-pSB1C3-(3)</td> | ||
+ | <td>15</td> | ||
+ | <td>2</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>2</td> | ||
+ | <td>H</td> | ||
+ | <td>6</td> | ||
+ | <td>6</td> | ||
+ | <td>51.5</td> | ||
+ | <td>82.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/15 AT-pSB1C3-(2)</td> | ||
+ | <td>19</td> | ||
+ | <td>-</td> | ||
+ | <td>2</td> | ||
+ | <td>2</td> | ||
+ | <td>-</td> | ||
+ | <td>MC</td> | ||
+ | <td>6</td> | ||
+ | <td>6</td> | ||
+ | <td>45</td> | ||
+ | <td>80</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/9 T7-RBS-pSB1C3</td> | ||
+ | <td>14</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>2</td> | ||
+ | <td>2</td> | ||
+ | <td>B</td> | ||
+ | <td>6</td> | ||
+ | <td>6</td> | ||
+ | <td>50</td> | ||
+ | <td>80</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/18 T7-RBS-AT-His-pSB1C3</td> | ||
+ | <td>33</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>3</td> | ||
+ | <td>3</td> | ||
+ | <td>B</td> | ||
+ | <td>9</td> | ||
+ | <td>9</td> | ||
+ | <td>63</td> | ||
+ | <td>120</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>UV-spequtrum measurement</h4> | ||
+ | <span class="kyoto-author">Yasuda and Murata</span> | ||
+ | |||
+ | Ethanenitrile solution of saturated MTOB (x10) | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2014/thumb/9/9c/Kyoto-Img087.jpg/362px-Kyoto-Img087.jpg"> | ||
+ | |||
+ | |||
+ | <h4>Mutation PCR</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <p>SAMmt (SpeI)</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Prime STAR MAX Premix(2x)</th> | ||
+ | <th>Fw SAMmt SpeI mutation primer</th> | ||
+ | <th>Rv SAMmt SpeI mutation primer</th> | ||
+ | <th>9/18 SAMmt-pSB1C3-4</th> | ||
+ | <th>MilliQ</th> | ||
+ | <th>Total</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>25µL</td> | ||
+ | <td>1µL</td> | ||
+ | <td>1µL</td> | ||
+ | <td>pick upµL</td> | ||
+ | <td>23µL</td> | ||
+ | <td>50µL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>98°C</td> | ||
+ | <td>98°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>72°C</td> | ||
+ | <td>12°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>15sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>Infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | |||
+ | |||
+ | <!--ここから先in藤田lab--> | ||
+ | |||
+ | <h4>Quality check of preligation</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | |||
+ | <table> | ||
+ | <tr><th>Sample</th><th>Concentration(ng/µL)</th><th>A260</th><th>A280</th><th>260/280</th><th>260/230</th></tr> | ||
+ | |||
+ | <tr><td>DiDECARB</td><td>538.36</td><td>10.767</td><td>5.734</td><td>1.88</td><td>2.07</td></tr> | ||
+ | <tr><td>DECARB</td><td>223.37</td><td>4.467</td><td>2.361</td><td>1.89</td><td>1.99</td></tr> | ||
+ | <tr><td>SAMmt-pSB1C3</td><td>185.24</td><td>3.705</td><td>1.896</td><td>1.95</td><td>1.87</td></tr> | ||
+ | <tr><td>REDOX</td><td>454.07</td><td>9.081</td><td>4.800</td><td>1.89</td><td>2.17</td></tr> | ||
+ | <tr><td>AT-pSB1C3</td><td>299.73</td><td>5.995</td><td>2.993</td><td>2.00</td><td>1.99</td></tr> | ||
+ | <tr><td>T7-RBS</td><td>306.31</td><td>6.126</td><td>2.901</td><td>2.11</td><td>1.97</td></tr> | ||
+ | <tr><td>T7-RBS-His</td><td>307.59</td><td>6.152</td><td>3.129</td><td>1.97</td><td>1.50</td></tr> | ||
+ | <tr><td>Pcon-pSB1C3</td><td>245.88</td><td>4.918</td><td>2.423</td><td>2.03</td><td>1.65</td></tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | |||
+ | <h4>PCR</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>DiDECARB</th> | ||
+ | <th>10x Buffer</th> | ||
+ | <th>2mM dNTPs</th> | ||
+ | <th>25mM MgSO4</th> | ||
+ | <th>#1</th> | ||
+ | <th>#2</th> | ||
+ | <th>KOD(1U/µL)</th> | ||
+ | <th>MilliQ</th> | ||
+ | <th>Total</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1µL</td> | ||
+ | <td>2.5µL</td> | ||
+ | <td>2.5µL</td> | ||
+ | <td>1.5µL</td> | ||
+ | <td>1.25µL</td> | ||
+ | <td>1.25µL</td> | ||
+ | <td>0.5µL</td> | ||
+ | <td>14.5µL</td> | ||
+ | <td>25</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>25µL x4 (100µL)</p> | ||
+ | |||
+ | <br> | ||
+ | <p> | ||
+ | (1)<br> | ||
+ | #1: DiDECARB OE-A (10 µM)<br> | ||
+ | #2: DiDECARB Mutation Rv (10 µM)<br> | ||
+ | </p> | ||
+ | |||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>98°C</td> | ||
+ | <td>50-65°C</td> | ||
+ | <td>68°C</td> | ||
+ | <td>15°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>48sec</td> | ||
+ | <td>Infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | |||
+ | |||
+ | <br> | ||
+ | <p> | ||
+ | (2)<br> | ||
+ | #1: DiDECARB OE-D (10 µM)<br> | ||
+ | #2: DiDECARB Mutation Fw (10 µM)<br> | ||
+ | </p> | ||
+ | <p> | ||
+ | (3)<br> | ||
+ | #1: RV-DiDECARB His (10 µM)<br> | ||
+ | #2: DiDECARB Mutation Fw (10 µM)<br> | ||
+ | </p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>98°C</td> | ||
+ | <td>50-65°C</td> | ||
+ | <td>68°C</td> | ||
+ | <td>15°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>78sec</td> | ||
+ | <td>Infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | |||
+ | <br> | ||
+ | <p> | ||
+ | (4)<br> | ||
+ | #1: DiDECARB OE-A (10 µM)<br> | ||
+ | #2: DiDECARB MutationP Rv (10 µM)<br> | ||
+ | </p> | ||
+ | <p> | ||
+ | (5)<br> | ||
+ | #1: DiDECARB MutationP-Fw (10 µM)<br> | ||
+ | #2: DiDECARB MutationE-Rv (10 µM)<br> | ||
+ | </p> | ||
+ | |||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>98°C</td> | ||
+ | <td>50-65°C</td> | ||
+ | <td>68°C</td> | ||
+ | <td>15°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>48sec</td> | ||
+ | <td>Infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | |||
+ | |||
+ | <br> | ||
+ | <p> | ||
+ | (6)<br> | ||
+ | #1: DiDECARB OE-D (10 µM)<br> | ||
+ | #2: DiDECARB Mutation-E-Fw (10 µM)<br> | ||
+ | </p> | ||
+ | <p> | ||
+ | (7)<br> | ||
+ | #1: RV-DiDECARB His (10 µM)<br> | ||
+ | #2: DiDECARB Mutation E-Fw (10 µM)<br> | ||
+ | </p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>98°C</td> | ||
+ | <td>50-65°C</td> | ||
+ | <td>68°C</td> | ||
+ | <td>15°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>78sec</td> | ||
+ | <td>Infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | |||
+ | <br> | ||
+ | <p> | ||
+ | (8)<br> | ||
+ | #1: SAMmt OE-A (10 µM)<br> | ||
+ | #2: SAMmt Mutation-Rv (10 µM)<br> | ||
+ | </p> | ||
+ | |||
+ | |||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>98°C</td> | ||
+ | <td>50-65°C</td> | ||
+ | <td>68°C</td> | ||
+ | <td>15°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>48sec</td> | ||
+ | <td>Infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | |||
+ | |||
+ | <br> | ||
+ | <p> | ||
+ | (9)<br> | ||
+ | #1: SAMmt OE-D (10 µM)<br> | ||
+ | #2: SAMmt Mutation-Fw (10 µM)<br> | ||
+ | </p> | ||
+ | <p> | ||
+ | (10)<br> | ||
+ | #1: RV-SAMmt His (10 µM)<br> | ||
+ | #2: SAMmt Mutation-Fw (10 µM)<br> | ||
+ | </p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>98°C</td> | ||
+ | <td>50-65°C</td> | ||
+ | <td>68°C</td> | ||
+ | <td>15°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>78sec</td> | ||
+ | <td>Infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | |||
+ | |||
+ | <br> | ||
+ | <p> | ||
+ | (11)<br> | ||
+ | #1: Fw-Pre-REDPX (10 µM)<br> | ||
+ | #2: Suf-REDOX-RV (10 µM)<br> | ||
+ | </p> | ||
+ | <p> | ||
+ | (12)<br> | ||
+ | #1: Fw-Pre-REDPX (10 µM)<br> | ||
+ | #2: RV-REDOX-His (10 µM)<br> | ||
+ | </p> | ||
+ | |||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>98°C</td> | ||
+ | <td>50-65°C</td> | ||
+ | <td>68°C</td> | ||
+ | <td>15°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>48sec</td> | ||
+ | <td>Infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | |||
+ | |||
+ | |||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | <table> | ||
+ | <tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th>XhoI</th><th>DraI</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | ||
+ | |||
+ | <tr><td>AT-pSB1C3 (299ng/µL)</td><td>4</td><td>-</td><td>1</td><td>-</td><td>1</td><td>1</td><td>1</td><td>M</td><td>5</td><td>5</td><td>32</td><td>50</td></tr> | ||
+ | |||
+ | <tr><td>AT-pSB1C3 (299ng/µL)</td><td>6.8</td><td>-</td><td>1</td><td>-</td><td>1</td><td>-</td><td>-</td><td>M</td><td>3</td><td>3</td><td>15.20</td><td>30</td></tr> | ||
+ | |||
+ | <tr><td>pSB1C3-T7-RBS (310ng/µL)</td><td>7</td><td>-</td><td>-</td><td>1</td><td>1</td><td>-</td><td>-</td><td>H</td><td>5</td><td>1*</td><td>35</td><td>50</td></tr> | ||
+ | |||
+ | <tr><td>Pcon-pSB1C3 (245ng/µL)</td><td>4</td><td>1</td><td>-</td><td>-</td><td>1</td><td>-</td><td>-</td><td>H</td><td>5</td><td>1*</td><td>38</td><td>50</td></tr> | ||
+ | |||
+ | |||
+ | </table> | ||
+ | |||
+ | <p>The cell marked * is not BSA but BAP.</p> | ||
+ | |||
+ | </div> | ||
+ | <div> | ||
+ | |||
+ | <a name="0919" class="kyoto-jump"></a> | ||
+ | <h3>9/19</h3> | ||
+ | |||
+ | <h4>Sequence PCR</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Number/(µL)</th> | ||
+ | <th>Sample</th> | ||
+ | <th>Template/(µL)</th> | ||
+ | <th>BigDye/(µL)</th> | ||
+ | <th>5x Buffer/(µL)</th> | ||
+ | <th colspan=2>Primer/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Conc/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>T7-RBS-AT-His(9/16-(2))</td> | ||
+ | <td>2.8</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VF</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.95</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>T7-RBS-AT-His(9/16-(3))</td> | ||
+ | <td>2.8</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VF</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.95</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>SAM-pSB1C3(4)</td> | ||
+ | <td>2.8</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VF</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.95</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>T7-RBS-AT-His(9/16-(2))</td> | ||
+ | <td>2.8</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VR</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.95</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>T7-RBS-AT-His(9/16-(3))</td> | ||
+ | <td>2.8</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VR</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.95</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td>SAM-pSB1C3(4)</td> | ||
+ | <td>2.8</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VR</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.95</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>7</td> | ||
+ | <td>T7-RBS-AT-His(9/16-(2))</td> | ||
+ | <td>2.8</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>seg-1</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.95</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>8</td> | ||
+ | <td>T7-RBS-AT-His(9/16-(3))</td> | ||
+ | <td>2.8</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>seg-1</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.95</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9</td> | ||
+ | <td>SAM-pSB1C3(4)</td> | ||
+ | <td>2.8</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>OE-B</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.95</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10</td> | ||
+ | <td>T7-RBS-AT-His(9/16-(2))</td> | ||
+ | <td>2.8</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>seg-2</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.95</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>11</td> | ||
+ | <td>9/23M PQLB</td> | ||
+ | <td>2.8</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>seg-2</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.95</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>96°C</td> | ||
+ | <td>96°C</td> | ||
+ | <td>50°C</td> | ||
+ | <td>60°C</td> | ||
+ | <td>4°C</td> | ||
+ | <td>4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>5sec</td> | ||
+ | <td>4min</td> | ||
+ | <td>1min</td> | ||
+ | <td>infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | |||
+ | <h4>Gel Extraction</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1,2,4,5,8</td> | ||
+ | <td>1kbp ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>T7-RBS-AT-His-pSB1C3 S,P</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td>T7-RBS-pSB1C3 S,P</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/7/7f/Kyoto-dms-0919_Gel_Extention_No1_before.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/4/4a/Kyoto-dms-0919_Gel_Extention_No1_after.jpg"> | ||
+ | |||
+ | <h4>Miniprep</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/18 T7-RBS-AT-His(2)</td> | ||
+ | <td>305</td> | ||
+ | <td>1.66</td> | ||
+ | <td>1.49</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/18 T7-RBS-His-pSB1C3(1)</td> | ||
+ | <td>280</td> | ||
+ | <td>1.68</td> | ||
+ | <td>1.67</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/18 REDOX(His)-YC Plac3(1)</td> | ||
+ | <td>985</td> | ||
+ | <td>1.74</td> | ||
+ | <td>2.39</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/18 REDOX(His)-YC Plac3(2)</td> | ||
+ | <td>1025</td> | ||
+ | <td>1.72</td> | ||
+ | <td>2.28</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Gel Extraction</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1,2</td> | ||
+ | <td>9/18 AT-pSB1C3 X,S</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>100bp ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5,6</td> | ||
+ | <td>9/18 AT-pSB1C3 E,P</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/3/36/Kyoto-dms-0919_Gel_Extention_No2_before.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/e/e2/Kyoto-dms-0919_Gel_Extention_No2_after.jpg"> | ||
+ | |||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(µL)</th> | ||
+ | <th>EcoR1/(µL)</th> | ||
+ | <th>Xba1/(µL)</th> | ||
+ | <th>Spe1/(µL)</th> | ||
+ | <th>Pst1/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th>BSA/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/19 T7-RBS-His Miniprep product</td> | ||
+ | <td>20</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>3</td> | ||
+ | <td>3</td> | ||
+ | <td>B</td> | ||
+ | <td>12</td> | ||
+ | <td>0</td> | ||
+ | <td>82</td> | ||
+ | <td>120</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/19 REDOX-3 M(1)</td> | ||
+ | <td>10.1</td> | ||
+ | <td>5</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>5</td> | ||
+ | <td>H</td> | ||
+ | <td>20</td> | ||
+ | <td>0</td> | ||
+ | <td>159.9</td> | ||
+ | <td>200</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/19 REDOX-3 M(1) control</td> | ||
+ | <td>0.1</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>H</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>8.9</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Electrophoresis</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>DiDECARB-OE-A 52.1°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>DiDECARB-OE-A 55.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>DiDECARB-OE-A 59.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>DiDECARB-OE-A 64.9°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>DiDECARB-OE-A 52.1°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td>DiDECARB-OE-A 55.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>7</td> | ||
+ | <td>DiDECARB-OE-A 59.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>8</td> | ||
+ | <td>DiDECARB-OE-A 64.9°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9</td> | ||
+ | <td>DiDECARB-Mutation PCR product Pst1 Fwd 52.1°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10</td> | ||
+ | <td>DiDECARB-Mutation PCR product Pst1 Fwd 55.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>11</td> | ||
+ | <td>DiDECARB-Mutation PCR product Pst1 Fwd 59.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>12</td> | ||
+ | <td>DiDECARB-Mutation PCR product Pst1 Fwd 64.9°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>13</td> | ||
+ | <td>REDOX-Fwd 52.1°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>14</td> | ||
+ | <td>REDOX-Fwd 55.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>15</td> | ||
+ | <td>REDOX-Fwd 59.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16</td> | ||
+ | <td>REDOX-Fwd 64.9°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>17</td> | ||
+ | <td>REDOX-Fwd-His 52.1°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>18</td> | ||
+ | <td>REDOX-Fwd-His 55.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>19</td> | ||
+ | <td>REDOX-Fwd-His 59.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>20</td> | ||
+ | <td>REDOX-Fwd-His 64.9°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>21</td> | ||
+ | <td>DiDECARB-Mutation PCR product Fwd-His 52.1°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>22</td> | ||
+ | <td>DiDECARB-Mutation PCR product Fwd-His 55.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>23</td> | ||
+ | <td>DiDECARB-Mutation PCR product Fwd-His 59.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>24</td> | ||
+ | <td>DiDECARB-Mutation PCR product Fwd-His 64.9°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>25</td> | ||
+ | <td>DiDECARB-Mutation PCR product Fwd 52.1°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>26</td> | ||
+ | <td>DiDECARB-Mutation PCR product Fwd 55.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>27</td> | ||
+ | <td>DiDECARB-Mutation PCR product Fwd 59.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>28</td> | ||
+ | <td>DiDECARB-Mutation PCR product Fwd 64.9°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>29</td> | ||
+ | <td>SAM-Mutation PCR product Fwd 52.1°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>30</td> | ||
+ | <td>SAM-Mutation PCR product Fwd 55.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>31</td> | ||
+ | <td>SAM-Mutation PCR product Fwd 59.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>32</td> | ||
+ | <td>SAM-Mutation PCR product Fwd 64.9°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>33</td> | ||
+ | <td>1kbp ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>34</td> | ||
+ | <td>SAM-Mutation PCR product Fwd 52.1°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>35</td> | ||
+ | <td>SAM-Mutation PCR product Fwd 55.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>36</td> | ||
+ | <td>SAM-Mutation PCR product Fwd 59.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>37</td> | ||
+ | <td>SAM-Mutation PCR product Fwd 64.9°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>38</td> | ||
+ | <td>SAM-Mutation PCR product Fwd-His 52.1°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>39</td> | ||
+ | <td>SAM-Mutation PCR product Fwd-His 55.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>40</td> | ||
+ | <td>SAM-Mutation PCR product Fwd-His 59.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>41</td> | ||
+ | <td>SAM-Mutation PCR product Fwd-His 64.9°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>42</td> | ||
+ | <td>1kbp ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>43</td> | ||
+ | <td>DiDECARB-Mutation PCR product E-His 52.1°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>44</td> | ||
+ | <td>DiDECARB-Mutation PCR product E-His 55.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>45</td> | ||
+ | <td>DiDECARB-Mutation PCR product E-His 59.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>46</td> | ||
+ | <td>DiDECARB-Mutation PCR product E-His 64.9°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>47</td> | ||
+ | <td>DiDECARB-Mutation PCR product E-Fwd 52.1°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>48</td> | ||
+ | <td>DiDECARB-Mutation PCR product E-Fwd 55.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>49</td> | ||
+ | <td>DiDECARB-Mutation PCR product E-Fwd 59.4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>50</td> | ||
+ | <td>DiDECARB-Mutation PCR product E-Fwd 64.9°C</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/d/d3/Kyoto-dms-0919_electrophoresis_No1.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/2/29/Kyoto-dms-0919_electrophoresis_No2.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/e/e1/Kyoto-dms-0919_electrophoresis_No3.jpg"> | ||
+ | |||
+ | <h4>PCR</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | <p>Template;DiDECARB-OE-A,DiDECARB Mutation product Fw</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>10x Buffer/(µL)</th> | ||
+ | <th>2.0mM dNTP/(µL)</th> | ||
+ | <th>25.mM MgSO4/(µL)</th> | ||
+ | <th>10.0µM DiDECARB-OE-A/(µL)</th> | ||
+ | <th>10.0µM DiDECARB-OE-D/(µL)</th> | ||
+ | <th>1.0U/µL KOD/(µL)</th> | ||
+ | <th>H2O</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10</td> | ||
+ | <td>10</td> | ||
+ | <td>6</td> | ||
+ | <td>5</td> | ||
+ | <td>5</td> | ||
+ | <td>2</td> | ||
+ | <td>up tp 100</td> | ||
+ | <td>100</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Template;DiDECARB-OE-A,DiDECARB Mutation product Fw-His</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>10x Buffer/(µL)</th> | ||
+ | <th>2.0mM dNTP/(µL)</th> | ||
+ | <th>25.mM MgSO4/(µL)</th> | ||
+ | <th>10.0µM DiDECARB-OE-A/(µL)</th> | ||
+ | <th>10.0µM Rv DiDECARB-His/(µL)</th> | ||
+ | <th>1.0U/µL KOD/(µL)</th> | ||
+ | <th>H2O</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10</td> | ||
+ | <td>10</td> | ||
+ | <td>6</td> | ||
+ | <td>5</td> | ||
+ | <td>5</td> | ||
+ | <td>2</td> | ||
+ | <td>up tp 100</td> | ||
+ | <td>100</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Template;DiDECARB-OE-A,DiDECARB Mutation produc Pst1t Fw,DiDECARB Mutation produc Eco1</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>10x Buffer/(µL)</th> | ||
+ | <th>2.0mM dNTP/(µL)</th> | ||
+ | <th>25.mM MgSO4/(µL)</th> | ||
+ | <th>10.0µM DiDECARB-OE-A/(µL)</th> | ||
+ | <th>10.0µM DiDECARB-OE-D/(µL)</th> | ||
+ | <th>1.0U/µL KOD/(µL)</th> | ||
+ | <th>H2O</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10</td> | ||
+ | <td>10</td> | ||
+ | <td>6</td> | ||
+ | <td>5</td> | ||
+ | <td>5</td> | ||
+ | <td>2</td> | ||
+ | <td>up tp 100</td> | ||
+ | <td>100</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Template;DiDECARB-OE-A,DiDECARB Mutation product Pst1 Fw,DiDECARB Mutation product Eco1-His</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>10x Buffer/(µL)</th> | ||
+ | <th>2.0mM dNTP/(µL)</th> | ||
+ | <th>25.mM MgSO4/(µL)</th> | ||
+ | <th>10.0µM DiDECARB-OE-A/(µL)</th> | ||
+ | <th>10.0µM Rv DiDECARB-His/(µL)</th> | ||
+ | <th>1.0U/µL KOD/(µL)</th> | ||
+ | <th>H2O</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10</td> | ||
+ | <td>10</td> | ||
+ | <td>6</td> | ||
+ | <td>5</td> | ||
+ | <td>5</td> | ||
+ | <td>2</td> | ||
+ | <td>up tp 100</td> | ||
+ | <td>100</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Template;SAM-OE-A,SAM Mutation product Fw</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>10x Buffer/(µL)</th> | ||
+ | <th>2.0mM dNTP/(µL)</th> | ||
+ | <th>25.mM MgSO4/(µL)</th> | ||
+ | <th>10.0µM SAM-OE-A/(µL)</th> | ||
+ | <th>10.0µM SAM-OE-D/(µL)</th> | ||
+ | <th>1.0U/µL KOD/(µL)</th> | ||
+ | <th>H2O</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10</td> | ||
+ | <td>10</td> | ||
+ | <td>6</td> | ||
+ | <td>5</td> | ||
+ | <td>5</td> | ||
+ | <td>2</td> | ||
+ | <td>up tp 100</td> | ||
+ | <td>100</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Template;SAM-OE-A,SAM Mutation product Fw-His</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>10x Buffer/(µL)</th> | ||
+ | <th>2.0mM dNTP/(µL)</th> | ||
+ | <th>25.mM MgSO4/(µL)</th> | ||
+ | <th>10.0µM SAM-OE-A/(µL)</th> | ||
+ | <th>10.0µM SAM His/(µL)</th> | ||
+ | <th>1.0U/µL KOD/(µL)</th> | ||
+ | <th>H2O</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10</td> | ||
+ | <td>10</td> | ||
+ | <td>6</td> | ||
+ | <td>5</td> | ||
+ | <td>5</td> | ||
+ | <td>2</td> | ||
+ | <td>up tp 100</td> | ||
+ | <td>100</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pSB1C3-AT/Pst1,Xba1,Xhol1,Dra1</td> | ||
+ | <td>24.75</td> | ||
+ | <td>1.90</td> | ||
+ | <td>1.92</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7RBS/Spe1,Pst1</td> | ||
+ | <td>8.48</td> | ||
+ | <td>1.91</td> | ||
+ | <td>1.01</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7RBS-His/Spe1,Pst1</td> | ||
+ | <td>35.98</td> | ||
+ | <td>1.88</td> | ||
+ | <td>1.78</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2014/6/62/Kyoto-dms-0919_electrophoresis_No4.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/9/96/Kyoto-dms-0919_electrophoresis_No5.jpg"> | ||
+ | |||
+ | <h4>Gel Extraction/Electrophoresis</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2~5</td> | ||
+ | <td>DiDECARB His</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6~8,10</td> | ||
+ | <td>DiDECARB</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>11~14</td> | ||
+ | <td>SAM</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16~20</td> | ||
+ | <td>SAM His</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>21~23,25</td> | ||
+ | <td>DE His</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>26~30</td> | ||
+ | <td>DE</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1,9,15,24</td> | ||
+ | <td>Blank</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>DNA Purification</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | <p>DiDECARB His</p> | ||
+ | <p>DiDECARB</p> | ||
+ | <p>SAM</p> | ||
+ | <p>SAM His</p> | ||
+ | |||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(ng)</th> | ||
+ | <th>EcoR1/(µL)</th> | ||
+ | <th>Xba1/(µL)</th> | ||
+ | <th>Spe1/(µL)</th> | ||
+ | <th>Pst1/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th>BSA/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DiDECARB His X,P</td> | ||
+ | <td>2000</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>M</td> | ||
+ | <td>3</td> | ||
+ | <td>3</td> | ||
+ | <td>up tp 30</td> | ||
+ | <td>30</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DiDECARB X,P</td> | ||
+ | <td>2000</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>M</td> | ||
+ | <td>3</td> | ||
+ | <td>3</td> | ||
+ | <td>up to 30</td> | ||
+ | <td>30</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DiDECARB E,P</td> | ||
+ | <td>2000</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>H</td> | ||
+ | <td>3</td> | ||
+ | <td>-</td> | ||
+ | <td>up to 30</td> | ||
+ | <td>30</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SAM X,P</td> | ||
+ | <td>2000</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>M</td> | ||
+ | <td>3</td> | ||
+ | <td>3</td> | ||
+ | <td>-</td> | ||
+ | <td>30</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SAM E,P</td> | ||
+ | <td>2000</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>H</td> | ||
+ | <td>3</td> | ||
+ | <td>-</td> | ||
+ | <td>up to 30</td> | ||
+ | <td>30</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SAM His X,P</td> | ||
+ | <td>2000</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>M</td> | ||
+ | <td>3</td> | ||
+ | <td>3</td> | ||
+ | <td>up to 30</td> | ||
+ | <td>30</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | </div> | ||
+ | <div> | ||
+ | |||
+ | <a name="0920" class="kyoto-jump"></a> | ||
+ | <h3>9/20</h3> | ||
+ | |||
+ | <h4>Electrophoresis</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>SAM His X,P</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>Di X,P</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>Di His X,P</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>SAM E,P</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>SAM X,P</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td>Di E,P</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>7</td> | ||
+ | <td>AT X,P 2</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>AT X,P 3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>T7-RBS S,P</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>dddD Mutation PCR product (2)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>dddD Mutation PCR product (1)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>T7-RBS pSB1C3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td>REDOX PCR product</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>7</td> | ||
+ | <td>REDOX His PCR product</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/9/9d/Kyoto-dms-0920_electrophoresis_No1.jpg"> | ||
+ | |||
+ | <h4>Transformation</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(µL)</th> | ||
+ | <th colspan=2>CompetentCells/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | <th>Medium</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>7-2 DiDECARB T7-RBS</td> | ||
+ | <td>2</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>10</td> | ||
+ | <td>12</td> | ||
+ | <td>SOC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>7-5 SAMmt T7-RBS</td> | ||
+ | <td>2</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>10</td> | ||
+ | <td>12</td> | ||
+ | <td>SOC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>7-11 T7-RBS dddD(1) Mutation PCR product</td> | ||
+ | <td>2</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>10</td> | ||
+ | <td>12</td> | ||
+ | <td>SOC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>7-12 T7-RBS dddD(3) Mutation PCR product</td> | ||
+ | <td>2</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>10</td> | ||
+ | <td>12</td> | ||
+ | <td>SOC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>7-13 T7-RBS AT</td> | ||
+ | <td>2</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>10</td> | ||
+ | <td>12</td> | ||
+ | <td>SOC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7-RBS control</td> | ||
+ | <td>2</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>10</td> | ||
+ | <td>12</td> | ||
+ | <td>SOC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10-1 DiDECARB pSB1C3</td> | ||
+ | <td>2</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>10</td> | ||
+ | <td>12</td> | ||
+ | <td>SOC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10-4 SAMmt pSB1C3</td> | ||
+ | <td>2</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>10</td> | ||
+ | <td>12</td> | ||
+ | <td>SOC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10-8 REDOX pSB1C3</td> | ||
+ | <td>2</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>10</td> | ||
+ | <td>12</td> | ||
+ | <td>SOC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pSB1C3 control</td> | ||
+ | <td>2</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>10</td> | ||
+ | <td>12</td> | ||
+ | <td>SOC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>14-3 T7-RBS SAM-His</td> | ||
+ | <td>2</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>10</td> | ||
+ | <td>12</td> | ||
+ | <td>SOC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7-RBS DiDECARB His</td> | ||
+ | <td>2</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>10</td> | ||
+ | <td>12</td> | ||
+ | <td>SOC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>REDOX T7 RBS His(14)-9</td> | ||
+ | <td>2</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>10</td> | ||
+ | <td>12</td> | ||
+ | <td>SOC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7-RBS-His control</td> | ||
+ | <td>2</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>10</td> | ||
+ | <td>12</td> | ||
+ | <td>SOC</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <div> | ||
+ | |||
+ | <a name="0922" class="kyoto-jump"></a> | ||
+ | <h3>9/22</h3> | ||
+ | |||
+ | <h4>Colony PCR</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Quick Tag/(µL)</th> | ||
+ | <th>VF2/(µL)</th> | ||
+ | <th>VR/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>0.2</td> | ||
+ | <td>0.2</td> | ||
+ | <td>4.6</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>94°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>68°C</td> | ||
+ | <td>12°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>30sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>150sec</td> | ||
+ | <td>infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <td>Number</td> | ||
+ | <td>Sample</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>1~8</th> | ||
+ | <th>REDOX-T7-RBS-His</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9~16</td> | ||
+ | <td>T7-RBS-SAM-His</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>17~24</td> | ||
+ | <td>T7-RBS control</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>25~32</td> | ||
+ | <td>T7-RBS-dddD</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>33~40</td> | ||
+ | <td>DiDECARB-pSB1C3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>41~48</td> | ||
+ | <td>pSB1C3 control</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>49~56</td> | ||
+ | <td>T7-RBS-DiDECARB</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>57~64</td> | ||
+ | <td>T7-RBS-His</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>65~72</td> | ||
+ | <td>REDOX-pSB1C3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>73~80</td> | ||
+ | <td>DiDECARB-T7-RBS</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>81~88</td> | ||
+ | <td>SAM-pSB1C3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>89~96</td> | ||
+ | <td>T7-RBS-sssS</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>97~104</td> | ||
+ | <td>T7-RBS-SAM</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>105</td> | ||
+ | <td>T7-RBS-AT</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>106~123</td> | ||
+ | <td>pBC345-AT</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/c/cf/Kyoto-dms-0922_colony_PCR_No1.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/a/a4/Kyoto-dms-0922_colony_PCR_No2.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/7/7a/Kyoto-dms-0922_colony_PCR_No3.jpg"> | ||
+ | <igm src="https://static.igem.org/mediawiki/2014/6/62/Kyoto-dms-0922_colony_PCR_No4.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/c/cc/Kyoto-dms-0922_colony_PCR_No5.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/4/49/Kyoto-dms-0922_colony_PCR_No6.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/5/51/Kyoto-dms-0922_colony_PCR_No7.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/8/87/Kyoto-dms-0922_colony_PCR_No8.jpg"> | ||
+ | |||
+ | <h4>Colony PCR</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <p>Master Mix</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Quick Tag/(µL)</th> | ||
+ | <th>VF2/(µL)</th> | ||
+ | <th>VR/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>225</td> | ||
+ | <td>9</td> | ||
+ | <td>9</td> | ||
+ | <td>217</td> | ||
+ | <td>450</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>94°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>68°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>30sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>150sec</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Number</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1~7</td> | ||
+ | <td>9/20 T7-RBS-REDOX-His</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>8~14</td> | ||
+ | <td>9/20 SAMmt-pSB1C3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>15~21</td> | ||
+ | <td>9/20 T7-RBS-SAMmt-His</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>22~28</td> | ||
+ | <td>9/20 DiDECARB-pSB1C3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>29~35</td> | ||
+ | <td>9/20 T7-RBS-DiDECARB</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>36~42</td> | ||
+ | <td>9/20 T7-RBS-DiDECARB-His</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>43</td> | ||
+ | <td>9/20 T7-RBS-dddD</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>44</td> | ||
+ | <td>9/20 DiDECARB-pSB1C3</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Miniprep</h4> | ||
+ | <span class="kyoto-author">Tatsui</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7-RBS-His</td> | ||
+ | <td>55</td> | ||
+ | <td>1.58</td> | ||
+ | <td>0.80</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Concentration Measurement</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7-RBS-His S,P</td> | ||
+ | <td>1.0</td> | ||
+ | <td>1.54</td> | ||
+ | <td>1.94</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7-RBS-His S,P</td> | ||
+ | <td>1.3</td> | ||
+ | <td>1.59</td> | ||
+ | <td>2.01</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7-RBS E,S</td> | ||
+ | <td>1.1</td> | ||
+ | <td>1.40</td> | ||
+ | <td>2.01</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>HT-pSB1C3 X,P</td> | ||
+ | <td>4.4</td> | ||
+ | <td>1.52</td> | ||
+ | <td>1.08</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>AT-pSB1C3 X,P</td> | ||
+ | <td>4.0</td> | ||
+ | <td>1.65</td> | ||
+ | <td>1.06</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Cut Check</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>Blank</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>100bp GeneRuler</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>T7 RBS-His S,P</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>T7-RBS S,P</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>T7-RBS E,P</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td>AT-pSB1C3 X,P</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>7</td> | ||
+ | <td>AT-pSB1C3 X,P</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/f/f8/Kyoto-dms-0922_cut_check_No1.jpg"> | ||
+ | |||
+ | <h4>Electrophoresis</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <p>(1)</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>100b ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2~8</td> | ||
+ | <td>T7-RBS-REDOX-His</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>(2)</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>100b ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2~8</td> | ||
+ | <td>SAMmt-pSB1C3</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>(3)</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>100b ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2~8</td> | ||
+ | <td>T7-RBS-SAMmt-His</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>(4)</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>100b ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2~8</td> | ||
+ | <td>DiDECARB-pSB1C3</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>(5)</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>100b ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2~8</td> | ||
+ | <td>T7-RBS-DiDECARB</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>(6)</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>100b ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2~8</td> | ||
+ | <td>T7-RBS-DiDECARB-His</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>(7)</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>Blank</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>Blank</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>100b ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>T7-RBS-dddD-1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>DiDECARB-pSB1C3-1</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/d/d8/Kyoto-dms-0922_electrophoresis_%28colony_PCR%29_No1.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/a/a6/Kyoto-dms-0922_electrophoresis_%28colony_PCR%29_No2.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/e/e1/Kyoto-dms-0922_electrophoresis_%28colony_PCR%29_No3.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/c/c0/Kyoto-dms-0922_electrophoresis_%28colony_PCR%29_No4.jpg"> | ||
+ | |||
+ | <h4>Liquid Culture</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Medium</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7-RBS-dddD</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Medium</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>REDOX-pSB1C3</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Medium</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7-RBS-SAMmt</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Colony PCR</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <p>Master Mix</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Quick Taq/(µL)</th> | ||
+ | <th>VF2/(µL)</th> | ||
+ | <th>VR/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>240</td> | ||
+ | <td>9.6</td> | ||
+ | <td>9.6</td> | ||
+ | <td>220.8</td> | ||
+ | <td>480</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>94°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>68°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>30sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>90sec</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Number</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1~16</td> | ||
+ | <td>T7-RBS-DiDECARB-His</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>19~32</td> | ||
+ | <td>T7-RBS-SMAmt-His</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>33~48</td> | ||
+ | <td>T7-RBS-REDOX-His</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <div> | ||
+ | |||
+ | <a name="0923" class="kyoto-jump"></a> | ||
+ | <h3>9/23</h3> | ||
+ | |||
+ | <h4>Miniprep</h4> | ||
+ | <span class="kyoto-author">Miniprep</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pB-22</td> | ||
+ | <td>250</td> | ||
+ | <td>1.61</td> | ||
+ | <td>1.69</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pB-23</td> | ||
+ | <td>125</td> | ||
+ | <td>2.07</td> | ||
+ | <td>2.90</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pQ-3</td> | ||
+ | <td>185</td> | ||
+ | <td>1.68</td> | ||
+ | <td>1.87</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pQ-5</td> | ||
+ | <td>210</td> | ||
+ | <td>1.72</td> | ||
+ | <td>1.86</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pLQB</td> | ||
+ | <td>90</td> | ||
+ | <td>2.40</td> | ||
+ | <td>3.09</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Electrophoresis</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <p>1</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Number</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>100bp ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2~8</td> | ||
+ | <td>T7-RBS-REDOX-His 2~7</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>2</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Number</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>100bp ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2~8</td> | ||
+ | <td>T7-RBS-REDOX-His 8~14</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>3</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Number</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>100bp ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2,3</td> | ||
+ | <td>T7-RBS-REDOX-His 15,16</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4~8</td> | ||
+ | <td>T7-RBS-SAMmt-His 1~5</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>4</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Number</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>100bp ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2~8</td> | ||
+ | <td>T7-RBS-SAMmt-His 6~12</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>5</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Number</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>100bp ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2~5</td> | ||
+ | <td>T7-RBS-SAMmt-His 13~16</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6~8</td> | ||
+ | <td>T7-RBS-DiDECARB-His 1~3</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>6</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Number</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>100bp ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2~8</td> | ||
+ | <td>T7-RBS-DiDECARB-His 4~10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>7</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Number</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>100bp ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2~7</td> | ||
+ | <td>T7-RBS-DiDECARB-His 11~16</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/5/5e/Kyoto-dms-0923_electrophoresis_No1.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/5/50/Kyoto-dms-0923_electrophoresis_No3.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/1/19/Kyoto-dms-0923_electrophoresis_No4.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/5/5d/Kyyoto-dms-0923_electrophoresis_No2.jpg"> | ||
+ | |||
+ | </div> | ||
+ | <div> | ||
+ | |||
+ | <a name="0924" class="kyoto-jump"></a> | ||
+ | <h3>9/24</h3> | ||
+ | |||
+ | <h4>Dephosphorylation</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <p>T7-RBS-His S,P</p> | ||
+ | |||
+ | <h4>Miniprep</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7-RBS-SAM 6</td> | ||
+ | <td>10.3</td> | ||
+ | <td>1.67</td> | ||
+ | <td>1.15</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/22 DiDECARB pSB1C3-4</td> | ||
+ | <td>11.3</td> | ||
+ | <td>1.54</td> | ||
+ | <td>1.11</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/22 DiDECARB pSB1C3-2</td> | ||
+ | <td>10.5</td> | ||
+ | <td>1.67</td> | ||
+ | <td>0.83</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/20 T7-RBS-dddD Mutation PCR product(3)-(4)</td> | ||
+ | <td>14.2</td> | ||
+ | <td>1.71</td> | ||
+ | <td>1.38</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>REDOX-pSB1C3(2)</td> | ||
+ | <td>9.8</td> | ||
+ | <td>1.71</td> | ||
+ | <td>0.83</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/20 T7-RBS-dddD Mutation PCR product(3)-(5)</td> | ||
+ | <td>12.7</td> | ||
+ | <td>1.66</td> | ||
+ | <td>1.42</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/22 DiDECARB pSB1C3-5</td> | ||
+ | <td>10.5</td> | ||
+ | <td>1.69</td> | ||
+ | <td>1.15</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Cycle PCR</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <p>Master Mix</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Number/(µL)</th> | ||
+ | <th>Sample</th> | ||
+ | <th>Template/(µL)</th> | ||
+ | <th>BigDye/(µL)</th> | ||
+ | <th>5x Buffer/(µL)</th> | ||
+ | <th colspan=2>Primer/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Conc/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>9/23M PQLB</td> | ||
+ | <td>4.4</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VF2</td> | ||
+ | <td>0.5</td> | ||
+ | <td>2.8</td> | ||
+ | <td>90</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>9/23M PQLB</td> | ||
+ | <td>4.4</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>pQLB seq-1</td> | ||
+ | <td>0.5</td> | ||
+ | <td>2.8</td> | ||
+ | <td>90</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>9/23M PQLB</td> | ||
+ | <td>4.4</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>pQLB seq-2</td> | ||
+ | <td>0.5</td> | ||
+ | <td>2.8</td> | ||
+ | <td>90</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>9/23M PQLB</td> | ||
+ | <td>4.4</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>pQLB seq-3</td> | ||
+ | <td>0.5</td> | ||
+ | <td>2.8</td> | ||
+ | <td>90</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>9/23M PQLB</td> | ||
+ | <td>4.4</td> | ||
+ | <td>0.5</td> | ||
+ | <td>pQLB seq-4</td> | ||
+ | <td>VF2</td> | ||
+ | <td>0.5</td> | ||
+ | <td>2.8</td> | ||
+ | <td>90</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td>9/23M PQLB</td> | ||
+ | <td>4.4</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VR</td> | ||
+ | <td>0.5</td> | ||
+ | <td>2.8</td> | ||
+ | <td>90</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>7</td> | ||
+ | <td>9/23M PQ-3</td> | ||
+ | <td>2.2</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VF2</td> | ||
+ | <td>0.5</td> | ||
+ | <td>5.0</td> | ||
+ | <td>185</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>8</td> | ||
+ | <td>9/23M PQ-3</td> | ||
+ | <td>2.2</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VR</td> | ||
+ | <td>0.5</td> | ||
+ | <td>5.0</td> | ||
+ | <td>185</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9</td> | ||
+ | <td>9/23M PQ-5</td> | ||
+ | <td>1.9</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VF2</td> | ||
+ | <td>0.5</td> | ||
+ | <td>5.3</td> | ||
+ | <td>210</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10</td> | ||
+ | <td>9/23M PQ-5</td> | ||
+ | <td>1.9</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VR</td> | ||
+ | <td>0.5</td> | ||
+ | <td>5.3</td> | ||
+ | <td>210</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>11</td> | ||
+ | <td>9/22M pB-23</td> | ||
+ | <td>3.2</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VF2</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.0</td> | ||
+ | <td>125</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>12</td> | ||
+ | <td>9/22M pB-23</td> | ||
+ | <td>3.2</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VR</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.0</td> | ||
+ | <td>125</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>13</td> | ||
+ | <td>9/23M pB-22</td> | ||
+ | <td>1.6</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VF2</td> | ||
+ | <td>0.5</td> | ||
+ | <td>5.7</td> | ||
+ | <td>250</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>14</td> | ||
+ | <td>9/23M pB-22</td> | ||
+ | <td>1.6</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VR</td> | ||
+ | <td>0.5</td> | ||
+ | <td>5.7</td> | ||
+ | <td>250</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>15</td> | ||
+ | <td>9/15 MP AT-pSB1C3 1</td> | ||
+ | <td>1.3</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VF2</td> | ||
+ | <td>0.5</td> | ||
+ | <td>6.0</td> | ||
+ | <td>299</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16</td> | ||
+ | <td>9/15 MP AT-pSB1C3 1</td> | ||
+ | <td>1.3</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>AT Seq1</td> | ||
+ | <td>0.5</td> | ||
+ | <td>6.0</td> | ||
+ | <td>299</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>17</td> | ||
+ | <td>9/15 MP AT-pSB1C3 1</td> | ||
+ | <td>1.3</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>AT Seq2</td> | ||
+ | <td>0.5</td> | ||
+ | <td>6.0</td> | ||
+ | <td>299</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>18</td> | ||
+ | <td>9/15 MP AT-pSB1C3 1</td> | ||
+ | <td>1.3</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VR</td> | ||
+ | <td>0.5</td> | ||
+ | <td>6.0</td> | ||
+ | <td>299</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>19</td> | ||
+ | <td>9/22 M REDOX pSB1C3</td> | ||
+ | <td>0.8</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VF2</td> | ||
+ | <td>0.5</td> | ||
+ | <td>6.4</td> | ||
+ | <td>490</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>20</td> | ||
+ | <td>9/22 M REDOX pSB1C3</td> | ||
+ | <td>0.8</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>REDOX Seq1</td> | ||
+ | <td>0.5</td> | ||
+ | <td>6.4</td> | ||
+ | <td>490</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>21</td> | ||
+ | <td>9/22 M REDOX pSB1C3</td> | ||
+ | <td>0.8</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VR</td> | ||
+ | <td>0.5</td> | ||
+ | <td>6.4</td> | ||
+ | <td>490</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>22</td> | ||
+ | <td>9/18 MP SAM-pSB1C3</td> | ||
+ | <td>2.1</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VF2</td> | ||
+ | <td>0.5</td> | ||
+ | <td>5.0</td> | ||
+ | <td>185</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>23</td> | ||
+ | <td>9/18 MP SAM-pSB1C3</td> | ||
+ | <td>2.1</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>SAM Seq1</td> | ||
+ | <td>0.5</td> | ||
+ | <td>5.0</td> | ||
+ | <td>185</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>24</td> | ||
+ | <td>9/18 MP SAM-pSB1C3</td> | ||
+ | <td>2.1</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VR</td> | ||
+ | <td>0.5</td> | ||
+ | <td>5.0</td> | ||
+ | <td>185</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>25</td> | ||
+ | <td>9/22 DiDECARB pSB1C3-4</td> | ||
+ | <td>0.7</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VF</td> | ||
+ | <td>0.5</td> | ||
+ | <td>6.5</td> | ||
+ | <td>565</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>26</td> | ||
+ | <td>9/22 DiDECARB pSB1C3-4</td> | ||
+ | <td>0.7</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>DiDECARB Seq1</td> | ||
+ | <td>0.5</td> | ||
+ | <td>6.5</td> | ||
+ | <td>565</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>27</td> | ||
+ | <td>9/22 DiDECARB pSB1C3-4</td> | ||
+ | <td>0.7</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>DiDECARB Seq2</td> | ||
+ | <td>0.5</td> | ||
+ | <td>6.5</td> | ||
+ | <td>565</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>28</td> | ||
+ | <td>9/22 DiDECARB pSB1C3-4</td> | ||
+ | <td>0.7</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>VR</td> | ||
+ | <td>0.5</td> | ||
+ | <td>6.5</td> | ||
+ | <td>565</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>DNA Purification</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/24 PCR product AT</td> | ||
+ | <td>265</td> | ||
+ | <td>1.67</td> | ||
+ | <td>2.10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(µL)</th> | ||
+ | <th>EcoR1/(µL)</th> | ||
+ | <th>Xba1/(µL)</th> | ||
+ | <th>Spe1/(µL)</th> | ||
+ | <th>Pst1/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th>BSA/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/24 REDOX(His) column</td> | ||
+ | <td>7.5</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>M</td> | ||
+ | <td>3</td> | ||
+ | <td>3</td> | ||
+ | <td>14.5</td> | ||
+ | <td>30</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | </div> | ||
+ | <div> | ||
+ | |||
+ | <a name="0925" class="kyoto-jump"></a> | ||
+ | <h3>9/25</h3> | ||
+ | |||
+ | <h4>Colony PCR</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Quick Taq/(µL)</th> | ||
+ | <th>VF2/(µL)</th> | ||
+ | <th>VR/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>0.2</td> | ||
+ | <td>0.2</td> | ||
+ | <td>4.6</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>94°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>68°C</td> | ||
+ | <td>12°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>30sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>1min</td> | ||
+ | <td>infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Number</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>T7-RBS-REDOX-His(1)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>T7-RBS-REDOX-His(2)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>T7-RBS-REDOX-His(3)</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/1/12/Kyoto-dms-0925_colony_PCR_No1.jpg"> | ||
+ | |||
+ | <h4>Electrophoresis</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1,2</td> | ||
+ | <td>Blank</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>100b ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4~6</td> | ||
+ | <td>T7-RBS-REDOX-His(1)~(3)</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Colony PCR</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <p>Master Mix</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Quick Taq/(µL)</th> | ||
+ | <th>VF2/(µL)</th> | ||
+ | <th>VR/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>130</td> | ||
+ | <td>5.2</td> | ||
+ | <td>5.2</td> | ||
+ | <td>119.6</td> | ||
+ | <td>260.0</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>94°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>68°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>30sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>90sec</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Number</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1~8</td> | ||
+ | <td>T7-RBS-REDOX-His (1)~(8)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9~16</td> | ||
+ | <td>T7-RBS-SAMmt-His (1)~(8)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>17~24</td> | ||
+ | <td>T7-RBS-DiDECARB-His (1)~(8)</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/d/d0/Kyoto-dms-0925_colony_PCR_No2.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/b/b5/Kyoto-dms-0925_colony_PCR_No3.jpg"> | ||
+ | |||
+ | <h4>Electrophoresis</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <p>1</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>100bp GeneRuler</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2~8</td> | ||
+ | <td>T7-RBS-REDOX-His(1)~(7)</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>2</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>100bp GeneRuler</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>T7-RBS-REDOX-His(8)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3~7</td> | ||
+ | <td>T7-RBS-SAMmt-His(1)~(5)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>8</td> | ||
+ | <td>Blank</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>3</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>100bp GeneRuler</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2~4</td> | ||
+ | <td>T7-RBS-SAMmt-His(6)~(8)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5~8</td> | ||
+ | <td>T7-RBS-DiDECARB-His(1)~(4)</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>4</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>100bp GeneRuler</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2,3,5,6</td> | ||
+ | <td>T7-RBS-DiDECARB-His(5)~(8)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>Blank</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Transformation</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(µL)</th> | ||
+ | <th colspan=2>CompetentCells/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | <th>Medium</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7-RBS-AT-His 9/19 M(3)</td> | ||
+ | <td>1</td> | ||
+ | <td>Zip BL21(DE3)</td> | ||
+ | <td>10</td> | ||
+ | <td>11</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7-RBS-dddD(n) 9/22 M (3)-5</td> | ||
+ | <td>1</td> | ||
+ | <td>Zip BL21(DE3)</td> | ||
+ | <td>10</td> | ||
+ | <td>11</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | </div> | ||
+ | <div> | ||
+ | |||
+ | <a name="0926" class="kyoto-jump"></a> | ||
+ | <h3>9/26</h3> | ||
+ | |||
+ | <h4>Colony PCR</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Quick Taq/(µL)</th> | ||
+ | <th>VF2/(µL)</th> | ||
+ | <th>VR/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>0.2</td> | ||
+ | <td>0.2</td> | ||
+ | <td>4.6</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>94°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>68°C</td> | ||
+ | <td>12°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5min</td> | ||
+ | <td>30sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>2min</td> | ||
+ | <td>infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Number</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1~8</td> | ||
+ | <td>T7-RBS-REDOX-His</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9~16</td> | ||
+ | <td>T7-RBS-SAMmt-His</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>17~24</td> | ||
+ | <td>T7-RBS-DiDECARB-His</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/e/e9/Kyoto-dms-0926_colony_PCR_No1.jpg"> | ||
+ | |||
+ | <h4>Electrophoresis</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1~8</td> | ||
+ | <td>T7-RBS-REDOX-His(1)~(8)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9~12,14~17</td> | ||
+ | <td>T7-RBS-SAMmt-His(1)~(8)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>18~25</td> | ||
+ | <td>T7-RBS-DiDECARB-His(1)~(8)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>13</td> | ||
+ | <td>100bp ladder</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div> | ||
+ | |||
+ | <a name="0927" class="kyoto-jump"></a> | ||
+ | <h3>9/27</h3> | ||
+ | |||
+ | <h4>Colony PCR</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Quick Taq/(µL)</th> | ||
+ | <th>VF2/(µL)</th> | ||
+ | <th>VR/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>0.2</td> | ||
+ | <td>0.2</td> | ||
+ | <td>4.6</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>94°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>68°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>30sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>160sec</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Number</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1~7</td> | ||
+ | <td>T7-RBS-REDOX-His</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>8~14</td> | ||
+ | <td>T7-RBS-SAMmt-His</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>15~20</td> | ||
+ | <td>T7-RBS-DiDECARB-His</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>21,22</td> | ||
+ | <td>T7-RBS-AT-His 9/25 Transformation(3),(4)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>23,24</td> | ||
+ | <td>T7-RBS-dddD 9/25 Transformation(3),(4)</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/b/b4/Kyoto-dms-0927_colony_PCR_No1.jpg"> | ||
+ | |||
+ | <h4>Cloning PCR</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <p>Master Mix</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>KOD Plus/(µL)</th> | ||
+ | <th>DNA CCAP 1023/11/(µL)</th> | ||
+ | <th>2mMdNTPs/(µL)</th> | ||
+ | <th>10xBuffer forKDDplusV2/(µL)</th> | ||
+ | <th>25mM MgSO4/(µL)</th> | ||
+ | <th>Fw AT cloning primer 61/(µL)</th> | ||
+ | <th>Rv AT cloning primer 61/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>2</td> | ||
+ | <td>5</td> | ||
+ | <td>5</td> | ||
+ | <td>3</td> | ||
+ | <td>1.5</td> | ||
+ | <td>1.5</td> | ||
+ | <td>31</td> | ||
+ | <td>50</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>98°C</td> | ||
+ | <td>56°C</td> | ||
+ | <td>68°C</td> | ||
+ | <td>12°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>90sec</td> | ||
+ | <td>infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | </div> | ||
+ | <div> | ||
+ | |||
+ | <a name="0928" class="kyoto-jump"></a> | ||
+ | <h3>9/28</h3> | ||
+ | |||
+ | <h4>OE-PCR 1st</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>KOD Plus/(µL)</th> | ||
+ | <th>9/27 AT PCR product/(µL)</th> | ||
+ | <th>10xBuffer for KODPlus/(µL)</th> | ||
+ | <th>2mMdNTPS/(µL)</th> | ||
+ | <th>25mM MgSO4/(µL)</th> | ||
+ | <th>Primer #1/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>pick up</td> | ||
+ | <td>5</td> | ||
+ | <td>5</td> | ||
+ | <td>3</td> | ||
+ | <td>1.5(each)</td> | ||
+ | <td>up to 50</td> | ||
+ | <td>50</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>1. #1 AT OE-A/AT OE-B</p> | ||
+ | <p>2. #1 AT OE-C/AT2 OE-B</p> | ||
+ | <p>3. #1 AT2 OE-C/AT OE-D</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Final Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>98°C</td> | ||
+ | <td>98°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>72°C</td> | ||
+ | <td>72°C</td> | ||
+ | <td>12°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>10sec</td> | ||
+ | <td>1min</td> | ||
+ | <td>5min</td> | ||
+ | <td>infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 25cycle</p> | ||
+ | |||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(µL)</th> | ||
+ | <th>EcoR1/(µL)</th> | ||
+ | <th>Xba1/(µL)</th> | ||
+ | <th>Spe1/(µL)</th> | ||
+ | <th>Pst1/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th>BSA/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>8/27 Miniprep product 1A2</td> | ||
+ | <td>7.6</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>3</td> | ||
+ | <td>3</td> | ||
+ | <td>B</td> | ||
+ | <td>12</td> | ||
+ | <td>1.2</td> | ||
+ | <td>93.2</td> | ||
+ | <td>120</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>control</td> | ||
+ | <td>0.5</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>B</td> | ||
+ | <td>1</td> | ||
+ | <td>0.1</td> | ||
+ | <td>8.4</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Gel Extraction</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>9/28 OE-(3)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>9/28 OE-(3)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>Blank</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>9/28 OE-(1)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>100bp ladder</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>9/28 OE-(3)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>9/28 OE-(2)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td>100bp ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2,3,5</td> | ||
+ | <td>Blank</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>OE-PCR 2nd</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <p>Master Mix</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>KOD Plus/(µL)</th> | ||
+ | <th>10xBuffer for KODPlus ver2/(µL)</th> | ||
+ | <th>2mMdNTPS/(µL)</th> | ||
+ | <th>25mM MgSO4/(µL)</th> | ||
+ | <th>Primer #1/(µL)</th> | ||
+ | <th>Template #2/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>5</td> | ||
+ | <td>5</td> | ||
+ | <td>3</td> | ||
+ | <td>2(each)</td> | ||
+ | <td>0.5(each)</td> | ||
+ | <td>up to 50</td> | ||
+ | <td>50</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>1. #1 AT OE-A/AT OE-D #2 9/28 OE-(1) gel extraction product/9/28 OE-(2) gel extraction product/9/28 OE-(3) gel extraction product</p> | ||
+ | <p>2. #1 AT OE-A/AT OE-D #2 9/27 AT PCR product 9/28 column</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Final Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>98°C</td> | ||
+ | <td>98°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>72°C</td> | ||
+ | <td>72°C</td> | ||
+ | <td>12°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>10sec</td> | ||
+ | <td>1min</td> | ||
+ | <td>5min</td> | ||
+ | <td>infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 25cycle</p> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/9/9c/Kyoto-dms-0928_OE-PCR_No1.jpg"> | ||
+ | |||
+ | </div> | ||
+ | <div> | ||
+ | |||
+ | <a name="0929" class="kyoto-jump"></a> | ||
+ | <h3>9/29</h3> | ||
+ | |||
+ | <h4>Mutation PCR</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>PrimeStar Max Premix/(µL)</th> | ||
+ | <th>SAMmt Mutation Fw/(µL)</th> | ||
+ | <th>SAMmt Mutation Rv/(µL)</th> | ||
+ | <th>9/18 MP SAM-pSB1C3/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>25</td> | ||
+ | <td>1</td> | ||
+ | <td>1</td> | ||
+ | <td>0.5</td> | ||
+ | <td>22.5</td> | ||
+ | <td>50</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>98°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>72°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10sec</td> | ||
+ | <td>15sec</td> | ||
+ | <td>55sec</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | |||
+ | <h4>Gel Extraction</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>100bp ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3,7</td> | ||
+ | <td>1A2 9/28 S,P</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9</td> | ||
+ | <td>9/28 Digestion control</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1,2,4,5,6,8</td> | ||
+ | <td>Blank</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(µL)</th> | ||
+ | <th>EcoR1/(µL)</th> | ||
+ | <th>Xba1/(µL)</th> | ||
+ | <th>Spe1/(µL)</th> | ||
+ | <th>Pst1/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th>BSA/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>8/27 M 1A2</td> | ||
+ | <td>7.6</td> | ||
+ | <td>-</td> | ||
+ | <td>3</td> | ||
+ | <td>-</td> | ||
+ | <td>3</td> | ||
+ | <td>D</td> | ||
+ | <td>12</td> | ||
+ | <td>1.2</td> | ||
+ | <td>93.2</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>control</td> | ||
+ | <td>0.5</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>D</td> | ||
+ | <td>1</td> | ||
+ | <td>0.1</td> | ||
+ | <td>8.4</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Gel Extraction</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>100bp ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>1A2 9/29 X,P</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td>9/29 Digestion control</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2,3,5</td> | ||
+ | <td>Blank</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/c/c8/Kyoto-dms-0929_Gel_Extraction_No1_before.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/3/39/Kyoto-dms-0929_Gel_Extraction_No1_after.jpg"> | ||
+ | |||
+ | <h4>Concentration Measurement</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1. 9/28 OE(1) 9/29 gel extraction product</td> | ||
+ | <td>17.9</td> | ||
+ | <td>1.92</td> | ||
+ | <td>0.94</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2. 9/28 OE(2) 9/29 gel extraction product</td> | ||
+ | <td>122.2</td> | ||
+ | <td>1.86</td> | ||
+ | <td>1.64</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3. 9/28 OE(3) 9/29 gel extraction product</td> | ||
+ | <td>78.8</td> | ||
+ | <td>1.89</td> | ||
+ | <td>1.67</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1A2 9/29 X,P</td> | ||
+ | <td>26.1</td> | ||
+ | <td>1.37</td> | ||
+ | <td>0.76</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Transformation</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(µL)</th> | ||
+ | <th colspan=2>CompetentCells/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | <th>Medium</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/29 Ligation DiDECARB(His)-1A2</td> | ||
+ | <td>1</td> | ||
+ | <td>Zip BL21(DE3)</td> | ||
+ | <td>10</td> | ||
+ | <td>11</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/29 Ligation REDOX(His)-1A2</td> | ||
+ | <td>1</td> | ||
+ | <td>Zip BL21(DE3)</td> | ||
+ | <td>10</td> | ||
+ | <td>11</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/29 SAMmt mutation</td> | ||
+ | <td>1</td> | ||
+ | <td>Zip BL21(DE3)</td> | ||
+ | <td>10</td> | ||
+ | <td>11</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <div> | ||
+ | |||
+ | <a name="0930" class="kyoto-jump"></a> | ||
+ | <h3>9/30</h3> | ||
+ | |||
+ | <h4>Mutation PCR</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>PrimeStar Max PreMix/(µL)</th> | ||
+ | <th>Template #1/(µL)</th> | ||
+ | <th>Primers #2/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>25</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1(each)</td> | ||
+ | <td>up to 50</td> | ||
+ | <td>50</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>1. #1 9/15 Miniprep AT-pSB1C3(1) #2 Fw:AT Mutation Insert/Rv:AT Mutation Insert</p> | ||
+ | <p>2. #1 9/19 Miniprep T7-RBS-AT(His) #2 Fw Add His to T7RBS/Rv Add His toT7 RBS</p> | ||
+ | <p>3. #1 9/13 Miniprep T7-RBS-pSB1C3 #2 Fw Add His to T7RBS/Rv Add His to T7RBS </p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>98°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>72°C</td> | ||
+ | <td>12°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10sec</td> | ||
+ | <td>15sec</td> | ||
+ | <td>20sec</td> | ||
+ | <td>infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 35cycle</p> | ||
+ | |||
+ | <h4>DNA Purification</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SAMmt-pSB1C3 9/30 column</td> | ||
+ | <td>136.3</td> | ||
+ | <td>1.87</td> | ||
+ | <td>1.68</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>OE-PCR 2nd</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <p>Master Mix</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>KOD Plus/(µL)</th> | ||
+ | <th>10xBuffer for KODPlus ver2/(µL)</th> | ||
+ | <th>2mMdNTPS/(µL)</th> | ||
+ | <th>25mM MgSO4/(µL)</th> | ||
+ | <th>Primer #1/(µL)</th> | ||
+ | <th>Template #2/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>5</td> | ||
+ | <td>5</td> | ||
+ | <td>3</td> | ||
+ | <td>2(each)</td> | ||
+ | <td>0.5(each)</td> | ||
+ | <td>up to 50</td> | ||
+ | <td>50</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>1. #1 AT OE-A/AT OE-D #2 9/28 OE-(1) gel extraction product/9/28 OE-(2) gel extraction product/9/28 OE-(3) gel extraction product</p> | ||
+ | <p>2. #1 AT OE-A/AT OE-D #2 9/27 AT PCR product 9/28 column</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Final Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>98°C</td> | ||
+ | <td>98°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>72°C</td> | ||
+ | <td>72°C</td> | ||
+ | <td>12°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>10sec</td> | ||
+ | <td>1min</td> | ||
+ | <td>5min</td> | ||
+ | <td>infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | |||
+ | <h4>Transformation</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(µL)</th> | ||
+ | <th colspan=2>CompetentCells/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | <th>Medium</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7-RBS-AT-His</td> | ||
+ | <td>1</td> | ||
+ | <td>self-making DH5α</td> | ||
+ | <td>10</td> | ||
+ | <td>11</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SAMmt-pSB1C3 9/29 Mutation PCR product</td> | ||
+ | <td>1</td> | ||
+ | <td>self-making DH5α</td> | ||
+ | <td>10</td> | ||
+ | <td>11</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>AT-pSB1C3 9/30 insertion Mutation PCR product</td> | ||
+ | <td>1</td> | ||
+ | <td>self-making DH5α</td> | ||
+ | <td>10</td> | ||
+ | <td>11</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7-RBS-His 9/30 Mutation PCR product</td> | ||
+ | <td>1</td> | ||
+ | <td>self-making DH5α</td> | ||
+ | <td>10</td> | ||
+ | <td>11</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>DNA Purification</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/30 OE-PCR 2nd product(Pre-AT-Suf)</td> | ||
+ | <td>217</td> | ||
+ | <td>1.85</td> | ||
+ | <td>1.22</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(µL)</th> | ||
+ | <th>EcoR1/(µL)</th> | ||
+ | <th>Xba1/(µL)</th> | ||
+ | <th>Spe1/(µL)</th> | ||
+ | <th>Pst1/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th>BSA/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Pre-At-Suf 9/30 Column</td> | ||
+ | <td>9.2</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>H</td> | ||
+ | <td>4</td> | ||
+ | <td>0.4</td> | ||
+ | <td>14.4</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | </div> | ||
+ | <div> | ||
+ | |||
+ | <a name="1001" class="kyoto-jump"></a> | ||
+ | <h3>10/1</h3> | ||
+ | |||
+ | <h4>DNA Purification</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>AT 9/30 E,P</td> | ||
+ | <td>38</td> | ||
+ | <td>1.84</td> | ||
+ | <td>0.46</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pSB1C3 E,P</td> | ||
+ | <td>5</td> | ||
+ | <td>1.78</td> | ||
+ | <td>0.27</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Transformation</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(µL)</th> | ||
+ | <th colspan=2>CompetentCells/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | <th>Medium</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/30 AT-pSB1C3</td> | ||
+ | <td>1</td> | ||
+ | <td>BL21(DE3)</td> | ||
+ | <td>10</td> | ||
+ | <td>11</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/30 T7-RBS-His</td> | ||
+ | <td>1</td> | ||
+ | <td>self-making DH5α 2/23</td> | ||
+ | <td>10</td> | ||
+ | <td>11</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/30 T7-RBS-AT-His</td> | ||
+ | <td>1</td> | ||
+ | <td>self-making DH5α 2/23</td> | ||
+ | <td>10</td> | ||
+ | <td>11</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/29 SAMmt-pSB1C3</td> | ||
+ | <td>1</td> | ||
+ | <td>self-making DH5α 2/23</td> | ||
+ | <td>10</td> | ||
+ | <td>11</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/22 T7-RBS-dddD(3)-5</td> | ||
+ | <td>1</td> | ||
+ | <td>self-making DH5α 2/23</td> | ||
+ | <td>10</td> | ||
+ | <td>11</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <div> | ||
+ | <a name="1002" class="kyoto-jump"></a> | ||
+ | <h3>10/2</h3> | ||
+ | <h4>Kination</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <p>Add His to T7RBS Fw</p> | ||
+ | <p>ATOE-D</p> | ||
+ | <h4>PCR</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>PrimeSTAR Premix/(µL)</th> | ||
+ | <th>Template #1/(ng)</th> | ||
+ | <th>Primer #2/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>50</td> | ||
+ | <td>400</td> | ||
+ | <td>2(each)</td> | ||
+ | <td>up to 100</td> | ||
+ | <td>100</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>#1 AT-pSB1C3(without (1))</p> | ||
+ | <p>#2 Fw:Add His to T7RBS/Rv:AT OE-D</p> | ||
+ | <h4>DNA Purification</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PCR product (1),(2) His-pSB1C3-AT</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PCR product(3) AT-pSB1C3 (AT Mota I)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PCR product(4) AT-pSB1C3 (AT Mota D)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PCR product(5) T7-RBS-His-pSB1C3</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Dpn1/(µL)</th> | ||
+ | <th>10/2 PCR AT-pSB1C3 miniprep product(3)/ | ||
+ | (µL)</th> | ||
+ | <th>10x Buffer(H)/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>8</td> | ||
+ | <td>5</td> | ||
+ | <td>36</td> | ||
+ | <td>50</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Electrophoresis</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>His pSB1C3-AT 10/2 DNA Purification</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>AT-pSB1C3 Mutation Deletion 10/2 DNA Purification</ | ||
+ | td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td>1kbp GeneRuler</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>7</td> | ||
+ | <td>AT-pSB1C3 Mutation Insert 10/2 DNA Purification</ | ||
+ | td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>8</td> | ||
+ | <td>T7-RBS AT His 10/2 DNA Purification</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1~3,9~12</td> | ||
+ | <td>Blank</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/thumb/2/24/IMG-1.jpg/791px-IMG-1.jpg"> | ||
+ | </div> | ||
+ | <div> | ||
+ | <a name="1003" class="kyoto-jump"></a> | ||
+ | <h3>10/3</h3> | ||
+ | <h4>Miniprep</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/14 AT-pSB1C3(1)</td> | ||
+ | <td>119</td> | ||
+ | <td>2.00</td> | ||
+ | <td>2.19</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/1 SAMmt(Mutated)-pSB1C3(1)</td> | ||
+ | <td>275</td> | ||
+ | <td>1.86</td> | ||
+ | <td>1.75</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/1 SAMmt(Mutated)-pSB1C3(2)</td> | ||
+ | <td>227</td> | ||
+ | <td>1.93</td> | ||
+ | <td>1.90</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/1 T7-RBS-His(1)</td> | ||
+ | <td>147</td> | ||
+ | <td>2.00</td> | ||
+ | <td>1.64</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/1 T7-RBS-His(2)</td> | ||
+ | <td>107</td> | ||
+ | <td>1.94</td> | ||
+ | <td>1.74</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Mutation PCR</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | <p>Master Mix</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>PrimeSTARPremix/(µL)</th> | ||
+ | <th>Template 10/3 Miniprep AT-pSB1C3(1)/(µL)</ | ||
+ | th> | ||
+ | <th>Primer AT Mutation Insert Rv/(µL)</th> | ||
+ | <th>AT Mutation Insert Fw/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>12.5</td> | ||
+ | <td>119</td> | ||
+ | <td>0.5</td> | ||
+ | <td>0.5</td> | ||
+ | <td>up to 25</td> | ||
+ | <td>25</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>68°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10sec</td> | ||
+ | <td>15sec</td> | ||
+ | <td>20sec</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 35cycle</p> | ||
+ | <h4>Gel Extraction</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>His-OE-(1)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>His-OE-(2)</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/thumb/e/ee/IMG_0001-1.jpg/606px-IMG_0001-1.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/thumb/e/e7/IMG_0001-2.jpg/774px-IMG_0001-2.jpg"> | ||
+ | <h4>Electrophoresis</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>AT-pSB1C3 Mutation insert 50.2°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>AT-pSB1C3 Mutation insert 52.8°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>AT-pSB1C3 Mutation insert 54.2°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>AT-pSB1C3 Mutation insert 55.7°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>AT-pSB1C3 Mutation insert 57.1°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td>10/2 DNA Purification His pSB1C3-AT</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>7</td> | ||
+ | <td>AT-pSB1C3 Mutation Deletion 50.2°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>8</td> | ||
+ | <td>AT-pSB1C3 Mutation Deletion 52.8°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9</td> | ||
+ | <td>AT-pSB1C3 Mutation Deletion 54.2°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10</td> | ||
+ | <td>AT-pSB1C3 Mutation Deletion 55.7°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>11</td> | ||
+ | <td>AT-pSB1C3 Mutation Deletion 57.1°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>12</td> | ||
+ | <td>100bp ladder</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/thumb/2/2a/IMG_0002-1.jpg/735px-IMG_0002-1.jpg"> | ||
+ | <h4>OE-PCR 1st</h4> | ||
+ | <span class="kyoto-author">Murata</span> | ||
+ | <p>(1)</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>KOD Plus/(µL)</th> | ||
+ | <th>10xBuffer for KOD Plus/(µL)</th> | ||
+ | <th>2mM dNTPs/(µL)</th> | ||
+ | <th>25mM MgSO4/(µL)</th> | ||
+ | <th>10/2 MP J04450(pSB1C3)/(µL)</th> | ||
+ | <th>VF2/(µL)</th> | ||
+ | <th>Rv mutation primer T7-RBS-His/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>5</td> | ||
+ | <td>5</td> | ||
+ | <td>3</td> | ||
+ | <td>1</td> | ||
+ | <td>1.5</td> | ||
+ | <td>1.5</td> | ||
+ | <td>32</td> | ||
+ | <td>50</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>(2)</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>KOD Plus/(µL)</th> | ||
+ | <th>10xBuffer for KOD Plus/(µL)</th> | ||
+ | <th>2mM dNTPs/(µL)</th> | ||
+ | <th>25mM MgSO4/(µL)</th> | ||
+ | <th>10/2 MP J04450(pSB1C3)/(µL)</th> | ||
+ | <th>Fw mutation primer T7-RBS-His/(µL)</th> | ||
+ | <th>Present from Santa/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>5</td> | ||
+ | <td>5</td> | ||
+ | <td>3</td> | ||
+ | <td>1</td> | ||
+ | <td>1.5</td> | ||
+ | <td>1.5</td> | ||
+ | <td>32</td> | ||
+ | <td>50</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>98°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>68°C</td> | ||
+ | <td>4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>3min</td> | ||
+ | <td>infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 35cycle</p> | ||
+ | <h4>OE-PCR 2nd</h4> | ||
+ | <span class="kyoto-author">Yausda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>KOD Plus/(µL)</th> | ||
+ | <th>10xBuffer for KOD Plus/(µL)</th> | ||
+ | <th>2mM dNTPs/(µL)</th> | ||
+ | <th>25mM MgSO4/(µL)</th> | ||
+ | <th>His OE-(1) 10/3 gel extraction product/ | ||
+ | (µL)</th> | ||
+ | <th>His OE-(2) 10/3 gel extraction product/ | ||
+ | (µL)</th> | ||
+ | <th>VF2/(µL)</th> | ||
+ | <th>Present from Santa/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>5</td> | ||
+ | <td>5</td> | ||
+ | <td>3</td> | ||
+ | <td>1</td> | ||
+ | <td>1</td> | ||
+ | <td>1.5</td> | ||
+ | <td>1.5</td> | ||
+ | <td>31</td> | ||
+ | <td>50</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>98°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>68°C</td> | ||
+ | <td>4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>3min</td> | ||
+ | <td>infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 35cycle</p> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/thumb/2/24/10042014gel-mk2.jpg/800px-10042014gel-mk2.jpg"> | ||
+ | |||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(µL)</th> | ||
+ | <th>Xba1/(µL)</th> | ||
+ | <th>ApnL1/(µL)</th> | ||
+ | <th>10XT/(µL)</th> | ||
+ | <th>BSA/(µL)</th> | ||
+ | <th>CIAP/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>AT-pSB1C3(1) 10/3 Miniprep</td> | ||
+ | <td>10</td> | ||
+ | <td>1</td> | ||
+ | <td>1</td> | ||
+ | <td>4</td> | ||
+ | <td>4</td> | ||
+ | <td>1</td> | ||
+ | <td>19</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>OE-PCR 2nd(T7 RBS His) without CIAP</td> | ||
+ | <td>10</td> | ||
+ | <td>1</td> | ||
+ | <td>1</td> | ||
+ | <td>4</td> | ||
+ | <td>4</td> | ||
+ | <td>-</td> | ||
+ | <td>20</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Ligation</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <p>A</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Vector/(µL)</th> | ||
+ | <th colspan=2>Insert/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th colspan=2>Ligase/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</tr> | ||
+ | <tr> | ||
+ | <td>pSB1C3 10/3 XA</td> | ||
+ | <td>2</td> | ||
+ | <td>T7 RBS His 10/3 XA</td> | ||
+ | <td>2</td> | ||
+ | <td>2xRapidLigBuffer</td> | ||
+ | <td>5</td> | ||
+ | <td>Promega T4 Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pSB1C3 10/3 XA</td> | ||
+ | <td>2</td> | ||
+ | <td>T7 RBS His 10/3 XA</td> | ||
+ | <td>-</td> | ||
+ | <td>2xRapidLigBuffer</td> | ||
+ | <td>5</td> | ||
+ | <td>Promega T4 Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>2</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>B</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Vector/(µL)</th> | ||
+ | <th colspan=2>Insert/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</tr> | ||
+ | <tr> | ||
+ | <td>pSB1C3 10/3 XA</td> | ||
+ | <td>2</td> | ||
+ | <td>T7 RBS His 10/3 XA</td> | ||
+ | <td>2</td> | ||
+ | <td>2xRapidLigBuffer</td> | ||
+ | <td>4</td> | ||
+ | <td>-</td> | ||
+ | <td>8</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pSB1C3 10/3 XA</td> | ||
+ | <td>1</td> | ||
+ | <td>T7 RBS His 10/3 XA</td> | ||
+ | <td>-</td> | ||
+ | <td>2xRapidLigBuffer</td> | ||
+ | <td>4</td> | ||
+ | <td>1</td> | ||
+ | <td>6</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Transformation</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>CompetentCells</th> | ||
+ | <th>Medium</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/3 Ligation products</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>DNA Purification</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/3 OE-PCR 2nd</td> | ||
+ | <td>204.5</td> | ||
+ | <td>1.82</td> | ||
+ | <td>2.33</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>DNA Purification</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/3 digestion product(1)</td> | ||
+ | <td>29.6</td> | ||
+ | <td>1.53</td> | ||
+ | <td>1.11</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/3 digestion product(2)</td> | ||
+ | <td>23.2</td> | ||
+ | <td>1.67</td> | ||
+ | <td>0.68</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/thumb/2/28/Kyoto0-Bio-Rad_2014-10-04_01hr_05min.jpg/800px-Kyoto0-Bio-Rad_2014-10-04_01hr_05min.jpg"> | ||
+ | <h4>DNA Purification</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>RFP OE-1</td> | ||
+ | <td>33.4</td> | ||
+ | <td>1.58</td> | ||
+ | <td>1.08</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>RFP OE-2</td> | ||
+ | <td>51.7</td> | ||
+ | <td>1.70</td> | ||
+ | <td>1.19</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/thumb/b/b8/Bio-Rad_2014-10-04_10hr_03min.jpg/800px-Bio-Rad_2014-10-04_10hr_03min.jpg"> | ||
+ | |||
+ | <h4>DNA Purification</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/3 OE-PCR 2nd</td> | ||
+ | <td>416.3</td> | ||
+ | <td>1.81</td> | ||
+ | <td>2.31</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Transformation</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>CompetentCells</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/4 Ligation product</td> | ||
+ | <td>DH5α</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div> | ||
+ | |||
+ | <a name="1004" class="kyoto-jump"></a> | ||
+ | <h3>10/4</h3> | ||
+ | <h4>Gel Extraction</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/4 Digestion product</td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/thumb/5/5c/Bio-Rad_2014-10-04_01hr_44min.jpg/800px-Bio-Rad_2014-10-04_01hr_44min.jpg"> | ||
+ | <h4>Ligation</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Vector/(µL)</th> | ||
+ | <th colspan=2>Insert/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th colspan=2>Ligase/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</tr> | ||
+ | <tr> | ||
+ | <td>pSB1C3 10/4 XA</td> | ||
+ | <td>2</td> | ||
+ | <td>RFP His 10/4 XA</td> | ||
+ | <td>2</td> | ||
+ | <td>2x Rapid Ligation Buffer</td> | ||
+ | <td>5</td> | ||
+ | <td>Promega T4 Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pSB1C3 10/4 XA</td> | ||
+ | <td>3</td> | ||
+ | <td>RFP His 10/4 XA</td> | ||
+ | <td>1</td> | ||
+ | <td>2x Rapid Ligation Buffer</td> | ||
+ | <td>5</td> | ||
+ | <td>Promega T4 Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pSB1C3 10/4 XA</td> | ||
+ | <td>1</td> | ||
+ | <td>RFP His 10/4 XA</td> | ||
+ | <td>3</td> | ||
+ | <td>2x Rapid Ligation Buffer</td> | ||
+ | <td>5</td> | ||
+ | <td>Promega T4 Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pSB1C3 10/4 XA</td> | ||
+ | <td>1</td> | ||
+ | <td>RFP His 10/4 XA</td> | ||
+ | <td>1</td> | ||
+ | <td>2x Rapid Ligation Buffer</td> | ||
+ | <td>5</td> | ||
+ | <td>Promega T4 Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>2</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | </div> | ||
+ | <div> | ||
+ | |||
+ | <a name="1005" class="kyoto-jump"></a> | ||
+ | <h3>10/5</h3> | ||
+ | <h4>OE-PCR 2nd</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>KOD Plus/(µL)</th> | ||
+ | <th>10xBuffer for KOD Plus/(µL)</th> | ||
+ | <th>2mM dNTPs/(µL)</th> | ||
+ | <th>25mM MgSO4/(µL)</th> | ||
+ | <th>Primers #1/(µL)</th> | ||
+ | <th>Templates #2/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>10</td> | ||
+ | <td>10</td> | ||
+ | <td>6</td> | ||
+ | <td>3(each)</td> | ||
+ | <td>2(each)</td> | ||
+ | <td>62</td> | ||
+ | <td>100</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>1. #1 VF2/Present from Santa #2 RFP OE(1)/RFP OE(2)</p> | ||
+ | <p>2. #1 VF2/Present from Santa #2 RFP OE(1)/His OE(2)</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Final Extension</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94°C</td> | ||
+ | <td>98°C</td> | ||
+ | <td>55°C</td> | ||
+ | <td>68°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>30sec</td> | ||
+ | <td>100min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 35cycle</p> | ||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(µL)</th> | ||
+ | <th>Xba1/(µL)</th> | ||
+ | <th>Spe1/(µL)</th> | ||
+ | <th>CIAP/(µL)</th> | ||
+ | <th>10XT/(µL)</th> | ||
+ | <th>BSA/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/5 RPFHis(1) 10/5 OE2(1)</td> | ||
+ | <td>5</td> | ||
+ | <td>1</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>4</td> | ||
+ | <td>4</td> | ||
+ | <td>20</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/5 RPFHis(2) 10/5 OE2(2)</td> | ||
+ | <td>5</td> | ||
+ | <td>1</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>4</td> | ||
+ | <td>4</td> | ||
+ | <td>20</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/5 RPFHis(3) 10/4 OE2 RPF His</td> | ||
+ | <td>5</td> | ||
+ | <td>1</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>4</td> | ||
+ | <td>4</td> | ||
+ | <td>20</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/5 pSB1C3 XA (1) 9/18 Mutated Bn-pSB1C310-3</td> | ||
+ | <td>5</td> | ||
+ | <td>1</td> | ||
+ | <td>1</td> | ||
+ | <td>1</td> | ||
+ | <td>4</td> | ||
+ | <td>4</td> | ||
+ | <td>19</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/5 pSB1C3 XA (2) 9/14 Mutated | ||
+ | dddD(mutated)-pSB1C3(2)</td> | ||
+ | <td>5</td> | ||
+ | <td>1</td> | ||
+ | <td>1</td> | ||
+ | <td>1</td> | ||
+ | <td>4</td> | ||
+ | <td>4</td> | ||
+ | <td>19</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Gel Extraction</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>(1)-1,(2)-6</td> | ||
+ | <td>SKY</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>(1)-2,3</td> | ||
+ | <td>12FP His 10/5 XA(1)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>(1)-4,5</td> | ||
+ | <td>pSB1C3 10/5 XA(2)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>(1)-6</td> | ||
+ | <td>RFP His 10/5 XA(2)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>(2)-1</td> | ||
+ | <td>RFP His 10/5 XA(2)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>(2)-2,3</td> | ||
+ | <td>RFP His 10/5 XA(3)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>(2)-4,5</td> | ||
+ | <td>pSB1C3 10/5 XA(1)</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>DNA Purification</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/5 RFP His1</td> | ||
+ | <td>205.9</td> | ||
+ | <td>1.87</td> | ||
+ | <td>2.39</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/5 RFP His2</td> | ||
+ | <td>157.6</td> | ||
+ | <td>1.84</td> | ||
+ | <td>2.34</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Ligation</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Vector/(µL)</th> | ||
+ | <th colspan=2>Insert/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th colspan=2>Ligase/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</tr> | ||
+ | <tr> | ||
+ | <td>pSB1C3 10/4 XA</td> | ||
+ | <td>1</td> | ||
+ | <td>RFP His 10/4 XA</td> | ||
+ | <td>1</td> | ||
+ | <td>TOYOBO Ligation High</td> | ||
+ | <td>2</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>RFP His 10/4 XA</td> | ||
+ | <td>1</td> | ||
+ | <td>TOYOBO Ligation High</td> | ||
+ | <td>2</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>4</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Transformation</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>CompetentCells</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/5 Ligation product</td> | ||
+ | <td>DH5α</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Liquid Culture</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/4 C1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/4 C2</td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <h4>Gel extraction</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>RFP His XA1</td> | ||
+ | <td>169.8</td> | ||
+ | <td>1.85</td> | ||
+ | <td>1.13</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>RFP His XA3</td> | ||
+ | <td>62.6</td> | ||
+ | <td>1.79</td> | ||
+ | <td>1.11</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pSB1C3 XA 2x</td> | ||
+ | <td>26.0</td> | ||
+ | <td>1.71</td> | ||
+ | <td>1.01</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | <div> | ||
+ | <a name="1006" class="kyoto-jump"></a> | ||
+ | <h3>10/6</h3> | ||
+ | <h4>Miniprep</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/5 Pcon RPF-His A</td> | ||
+ | <td>133</td> | ||
+ | <td>1.93</td> | ||
+ | <td>2.24</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/5 Pcon RPF-His B</td> | ||
+ | <td>133</td> | ||
+ | <td>1.90</td> | ||
+ | <td>1.83</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/5 Pcon RPF-His C</td> | ||
+ | <td>137</td> | ||
+ | <td>1.80</td> | ||
+ | <td>1.72</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/5 Pcon RPF-His D</td> | ||
+ | <td>142</td> | ||
+ | <td>1.98</td> | ||
+ | <td>2.61</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>PCR</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <p>10/6 Pcon RPFHisA VF</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Template/(µL)</th> | ||
+ | <th>Bigdye/(µL)</th> | ||
+ | <th>5xBuffer/(µL)</th> | ||
+ | <th>Primer/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2.9</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.85</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>10/6 Pcon RPFHisA VR</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Template/(µL)</th> | ||
+ | <th>Bigdye/(µL)</th> | ||
+ | <th>5xBuffer/(µL)</th> | ||
+ | <th>Primer/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2.9</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.85</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>10/6 Pcon RPFHisB VF</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Template/(µL)</th> | ||
+ | <th>Bigdye/(µL)</th> | ||
+ | <th>5xBuffer/(µL)</th> | ||
+ | <th>Primer/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3.0</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.75</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>10/6 Pcon RPFHisB VR</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Template/(µL)</th> | ||
+ | <th>Bigdye/(µL)</th> | ||
+ | <th>5xBuffer/(µL)</th> | ||
+ | <th>Primer/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3.0</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.75</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>10/6 Pcon RPFHisC VF</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Template/(µL)</th> | ||
+ | <th>Bigdye/(µL)</th> | ||
+ | <th>5xBuffer/(µL)</th> | ||
+ | <th>Primer/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2.9</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.85</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>10/6 Pcon RPFHisC VR</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Template/(µL)</th> | ||
+ | <th>Bigdye/(µL)</th> | ||
+ | <th>5xBuffer/(µL)</th> | ||
+ | <th>Primer/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2.9</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.85</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>10/6 Pcon RPFHisD VF</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Template/(µL)</th> | ||
+ | <th>Bigdye/(µL)</th> | ||
+ | <th>5xBuffer/(µL)</th> | ||
+ | <th>Primer/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2.8</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.95</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>10/6 Pcon RPFHisD VR</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Template/(µL)</th> | ||
+ | <th>Bigdye/(µL)</th> | ||
+ | <th>5xBuffer/(µL)</th> | ||
+ | <th>Primer/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2.8</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.95</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>96°C</td> | ||
+ | <td>96°C</td> | ||
+ | <td>50°C</td> | ||
+ | <td>60°C</td> | ||
+ | <td>4°C</td> | ||
+ | <td>4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>5sec</td> | ||
+ | <td>4min</td> | ||
+ | <td>1min</td> | ||
+ | <td>infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | </div> | ||
+ | <div> | ||
+ | |||
+ | <a name="1007" class="kyoto-jump"></a> | ||
+ | <h3>10/7</h3> | ||
+ | <h4>PCR</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <p>AT(1)</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Template/(µL)</th> | ||
+ | <th>Bigdye/(µL)</th> | ||
+ | <th>5xBuffer/(µL)</th> | ||
+ | <th>Primer/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4.0</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>0.5</td> | ||
+ | <td>3.25</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>AT(2)</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Template/(µL)</th> | ||
+ | <th>Bigdye/(µL)</th> | ||
+ | <th>5xBuffer/(µL)</th> | ||
+ | <th>Primer/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2.0</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>0.5</td> | ||
+ | <td>5.25</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>AT(3)</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Template/(µL)</th> | ||
+ | <th>Bigdye/(µL)</th> | ||
+ | <th>5xBuffer/(µL)</th> | ||
+ | <th>Primer/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2.0</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>0.5</td> | ||
+ | <td>5.25</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>AT(4)</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Template/(µL)</th> | ||
+ | <th>Bigdye/(µL)</th> | ||
+ | <th>5xBuffer/(µL)</th> | ||
+ | <th>Primer/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2.5</td> | ||
+ | <td>0.5</td> | ||
+ | <td>1.75</td> | ||
+ | <td>0.5</td> | ||
+ | <td>4.75</td> | ||
+ | <td>10.5</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>96°C</td> | ||
+ | <td>96°C</td> | ||
+ | <td>50°C</td> | ||
+ | <td>60°C</td> | ||
+ | <td>4°C</td> | ||
+ | <td>4°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2min</td> | ||
+ | <td>10sec</td> | ||
+ | <td>5sec</td> | ||
+ | <td>4min</td> | ||
+ | <td>1min</td> | ||
+ | <td>infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(µL)</th> | ||
+ | <th>EcoR1/(µL)</th> | ||
+ | <th>Xba1/(µL)</th> | ||
+ | <th>Spe1/(µL)</th> | ||
+ | <th>Pst1/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th>BSA/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/6 T7-RBS-His(2)</td> | ||
+ | <td>12</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>1</td> | ||
+ | <td>B</td> | ||
+ | <td>4</td> | ||
+ | <td>0.4</td> | ||
+ | <td>21.6</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/6 T7-RBS-His(2) control</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>B</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>8</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/6 T7-RBS-His(3)</td> | ||
+ | <td>20</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>2</td> | ||
+ | <td>2</td> | ||
+ | <td>B</td> | ||
+ | <td>8</td> | ||
+ | <td>0.8</td> | ||
+ | <td>47.2</td> | ||
+ | <td>80</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/6 T7-RBS-His(3) control</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>B</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>8</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Gel Extraction</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>T7RBSHis(3) 10/7 S,P control</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>T7RBSHis(3) 10/7 S,P Experimental</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>100bp ladder</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>Blank</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>7RBSHis(2) 10/7 S,P Experimental</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td>7RBSHis(2) 10/7 S,P control</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>T7RBSHis(3) 10/7 S,P Experimental</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1,2,4,5,6</td> | ||
+ | <td>Blank</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7RBSHis(3) low</td> | ||
+ | <td>38</td> | ||
+ | <td>1.52</td> | ||
+ | <td>1.30</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7RBSHis(2)</td> | ||
+ | <td>46</td> | ||
+ | <td>1.49</td> | ||
+ | <td>1.53</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7RBSHis(3) high</td> | ||
+ | <td>28</td> | ||
+ | <td>2.04</td> | ||
+ | <td>0.38</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | </div> | ||
+ | <div> | ||
+ | |||
+ | <a name="1008" class="kyoto-jump"></a> | ||
+ | <h3>10/8</h3> | ||
+ | <h4>Liquid Culture</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Medium</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BL21</td> | ||
+ | <td>LB</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Liquid Culture</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Medium</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/30 AT-pSB1C3 insertion mutated</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9/30 SAMmt-pSB1C3 mutated</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <div> | ||
+ | |||
+ | <a name="1010" class="kyoto-jump"></a> | ||
+ | <h3>10/10</h3> | ||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(µL)</th> | ||
+ | <th>EcoR1/(µL)</th> | ||
+ | <th>Xba1/(µL)</th> | ||
+ | <th>Spe1/(µL)</th> | ||
+ | <th>Pst1/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th>BSA/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>AT(Mutation Insert)-pSB1C3(2)</td> | ||
+ | <td>20</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>D</td> | ||
+ | <td>4</td> | ||
+ | <td>0.4</td> | ||
+ | <td>3.6</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Dephosphorylation</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>10/8 T7 RBSHis(2)S,P/(µL)</th> | ||
+ | <th>CIAP/(µL)</th> | ||
+ | <th>10xBuffer/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>12</td> | ||
+ | <td>1</td> | ||
+ | <td>2</td> | ||
+ | <td>5</td> | ||
+ | <td>20</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <td>37°C</td> | ||
+ | <td>50°C</td> | ||
+ | <td>15°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>30min</td> | ||
+ | <td>15min</td> | ||
+ | <td>30min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Miniprep</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Liquid Culture product | ||
+ | SAM(Mutated)-pSB1C3(1)~(4)</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Cloning PCR</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>2X KAPA HiFI HotStart ReadyMix/(µL)</th> | ||
+ | <th>AT OE-A/(µL)</th> | ||
+ | <th>AT cloning for His/(µL)</th> | ||
+ | <th>10/7 M AT(Mutation Insert)-pSB1C3(2)/(µL)</ | ||
+ | th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>12.5</td> | ||
+ | <td>0.75</td> | ||
+ | <td>0.75</td> | ||
+ | <td>up to 25</td> | ||
+ | <td>11</td> | ||
+ | <td>25</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Final Extension</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>95°C</td> | ||
+ | <td>98°C</td> | ||
+ | <td>60°C</td> | ||
+ | <td>72°C</td> | ||
+ | <td>72°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5min</td> | ||
+ | <td>20sec</td> | ||
+ | <td>15sec</td> | ||
+ | <td>90sec</td> | ||
+ | <td>5sec</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 35cycle</p> | ||
+ | <h4>Gel Extraction</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>AT pSB1C3 (Mutation Insert)(2)10/3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>Blank</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>100bp ladder</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(µL)</th> | ||
+ | <th>EcoR1/(µL)</th> | ||
+ | <th>Xba1/(µL)</th> | ||
+ | <th>Spe1/(µL)</th> | ||
+ | <th>Pst1/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th>BSA/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/10 Miniprep product | ||
+ | SAMmt(Mutated)-pSB1C3(1)</td> | ||
+ | <td>10</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>E</td> | ||
+ | <td>4</td> | ||
+ | <td>0.4</td> | ||
+ | <td>24.6</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/10 Miniprep product | ||
+ | SAMmt(Mutated)-pSB1C3(2)</td> | ||
+ | <td>10</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>E</td> | ||
+ | <td>4</td> | ||
+ | <td>0.4</td> | ||
+ | <td>24.6</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/10 Miniprep product | ||
+ | SAMmt(Mutated)-pSB1C3(3)</td> | ||
+ | <td>10</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>E</td> | ||
+ | <td>4</td> | ||
+ | <td>0.4</td> | ||
+ | <td>24.6</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/10 Miniprep product | ||
+ | SAMmt(Mutated)-pSB1C3(4)</td> | ||
+ | <td>10</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>E</td> | ||
+ | <td>4</td> | ||
+ | <td>0.4</td> | ||
+ | <td>24.6</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Ligation</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Vector/(µL)</th> | ||
+ | <th colspan=2>Insert/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th colspan=2>Ligase/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</tr> | ||
+ | <tr> | ||
+ | <td>T7 RBS-pSB1C3 S,P</td> | ||
+ | <td>2</td> | ||
+ | <td>10/10 gel extraction product(true)XP</td> | ||
+ | <td>2</td> | ||
+ | <td>2xRapidLigaseBuffer</td> | ||
+ | <td>5</td> | ||
+ | <td>Promega T4 Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7 RBS-pSB1C3 S,P</td> | ||
+ | <td>1</td> | ||
+ | <td>10/10 gel extraction product(true)XP</td> | ||
+ | <td>3</td> | ||
+ | <td>2xRapidLigaseBuffer</td> | ||
+ | <td>5</td> | ||
+ | <td>Promega T4 Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7 RBS-pSB1C3 S,P</td> | ||
+ | <td>2</td> | ||
+ | <td>10/10 gel extraction product(true)XP</td> | ||
+ | <td>0</td> | ||
+ | <td>2xRapidLigaseBuffer</td> | ||
+ | <td>5</td> | ||
+ | <td>Promega T4 Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>2</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7 RBS-pSB1C3 S,P</td> | ||
+ | <td>2</td> | ||
+ | <td>10/10 gel extraction product(true)XP</td> | ||
+ | <td>2</td> | ||
+ | <td>2xRapidLigaseBuffer</td> | ||
+ | <td>5</td> | ||
+ | <td>Promega T4 Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7 RBS-pSB1C3 S,P</td> | ||
+ | <td>1</td> | ||
+ | <td>10/10 gel extraction product(true)XP</td> | ||
+ | <td>3</td> | ||
+ | <td>2xRapidLigaseBuffer</td> | ||
+ | <td>5</td> | ||
+ | <td>Promega T4 Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7 RBS-pSB1C3 S,P</td> | ||
+ | <td>2</td> | ||
+ | <td>10/10 gel extraction product(true)XP</td> | ||
+ | <td>0</td> | ||
+ | <td>2xRapidLigaseBuffer</td> | ||
+ | <td>5</td> | ||
+ | <td>Promega T4 Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>2</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Transformation</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>CompetentCells</th> | ||
+ | <th>Medium</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/10 Ligation product</td> | ||
+ | <td>BL21(DE3)</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | </div> | ||
+ | <div> | ||
+ | |||
+ | <a name="1011" class="kyoto-jump"></a> | ||
+ | <h3>10/11</h3> | ||
+ | <h4>Miniprep</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7RBSHis(2)</td> | ||
+ | <td>217.4</td> | ||
+ | <td>1.92</td> | ||
+ | <td>2.16</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7RBSHis(3)</td> | ||
+ | <td>418.0</td> | ||
+ | <td>1.95</td> | ||
+ | <td>2.30</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>AT-pSB1C3(2)</td> | ||
+ | <td>258.0</td> | ||
+ | <td>1.96</td> | ||
+ | <td>2.25</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(µL)</th> | ||
+ | <th>EcoR1/(µL)</th> | ||
+ | <th>Xba1/(µL)</th> | ||
+ | <th>Spe1/(µL)</th> | ||
+ | <th>Pst1/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th>BSA/(µL)</th> | ||
+ | <th>CIAP/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/11 miniprep product T7RBSHis(2)</td> | ||
+ | <td>10</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>2</td> | ||
+ | <td>2</td> | ||
+ | <td>B</td> | ||
+ | <td>8</td> | ||
+ | <td>0.8</td> | ||
+ | <td>47.2</td> | ||
+ | <td>80</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/11 miniprep product T7RBSHis(3)</td> | ||
+ | <td>10</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>2</td> | ||
+ | <td>2</td> | ||
+ | <td>B</td> | ||
+ | <td>8</td> | ||
+ | <td>0.8</td> | ||
+ | <td>57.2</td> | ||
+ | <td>80</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/11 miniprep product AT-pSB1C3(2)</td> | ||
+ | <td>10</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>B</td> | ||
+ | <td>4</td> | ||
+ | <td>0.4</td> | ||
+ | <td>0</td> | ||
+ | <td>23.6</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/10 miniprep product SAM(mutated)-pSB1C3-(1)</ | ||
+ | td> | ||
+ | <td>10</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>B</td> | ||
+ | <td>4</td> | ||
+ | <td>0.4</td> | ||
+ | <td>0</td> | ||
+ | <td>24.6</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/10 miniprep product SAM(mutated)-pSB1C3-(2)</ | ||
+ | td> | ||
+ | <td>10</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>B</td> | ||
+ | <td>4</td> | ||
+ | <td>0.4</td> | ||
+ | <td>0</td> | ||
+ | <td>24.6</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/10 miniprep product SAM(mutated)-pSB1C3-(3)</ | ||
+ | td> | ||
+ | <td>10</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>B</td> | ||
+ | <td>4</td> | ||
+ | <td>0.4</td> | ||
+ | <td>0</td> | ||
+ | <td>24.6</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/10 miniprep product SAM(mutated)-pSB1C3-(4)</ | ||
+ | td> | ||
+ | <td>10</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>B</td> | ||
+ | <td>4</td> | ||
+ | <td>0.4</td> | ||
+ | <td>0</td> | ||
+ | <td>24.6</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>DNA Purification</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/10 PCR Cloning product AT</td> | ||
+ | <td>207.6</td> | ||
+ | <td>1.88</td> | ||
+ | <td>2.00</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(µL)</th> | ||
+ | <th>EcoR1/(µL)</th> | ||
+ | <th>Xba1/(µL)</th> | ||
+ | <th>Spe1/(µL)</th> | ||
+ | <th>Pst1/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th>BSA/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/11 DNA Purification product AT(His)</td> | ||
+ | <td>10</td> | ||
+ | <td>0</td> | ||
+ | <td>1</td> | ||
+ | <td>0</td> | ||
+ | <td>1</td> | ||
+ | <td>D</td> | ||
+ | <td>4</td> | ||
+ | <td>0.4</td> | ||
+ | <td>24.6</td> | ||
+ | <td>40</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h4>Transformation</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>CompetentCells</th> | ||
+ | <th>Medium</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/10 Ligation</td> | ||
+ | <td>BL21(DE3)</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | |||
+ | <div> | ||
+ | <a name="1013" class="kyoto-jump"></a> | ||
+ | <h3>10/13</h3> | ||
+ | |||
+ | |||
+ | <h4>Miniprep</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | |||
+ | <tr><td>1. 10/13 T7-RBS (1)</td><td>334.6</td><td>1.91</td><td>2.15</td></tr> | ||
+ | <tr><td>2. 10/13 T7-RBS (1)</td><td>319.6</td><td>1.87</td><td>1.72</td></tr> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | |||
+ | <h4>Electrophoresis</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr><th>Lane</th><th colspan=3>Sample</th></tr> | ||
+ | <tr><td>1</td><td>-</td></tr> | ||
+ | <tr><td>2</td><td>-</td></tr> | ||
+ | <tr><td>3</td><td>-</td></tr> | ||
+ | <tr><td>4</td><td>1kb ladder</td></tr> | ||
+ | <tr><td>5</td><td>10/11 SAMmt-pSB1C3 (S)-(2)</td></tr> | ||
+ | <tr><td>6</td><td>10/11 SAMmt-pSB1C3 (S)-(3)</td></tr> | ||
+ | <tr><td>7</td><td>10/11 SAMmt-pSB1C3 (S)-(1)</td></tr> | ||
+ | <tr><td>8</td><td>10/11 SAMmt-pSB1C3 (S)-(4)</td></tr> | ||
+ | <tr><td>9</td><td>1kb ladder</td></tr> | ||
+ | |||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/thumb/d/d2/IMG_0004.jpg/780px-IMG_0004.jpg"> | ||
+ | |||
+ | <h4>Gel Extraction</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr><th>Lane</th><th>Sample</th></tr> | ||
+ | <tr><td>1</td><td>-</td></tr> | ||
+ | <tr><td>2</td><td>-</td></tr> | ||
+ | <tr><td>3</td><td>10/11 AT-pSB1C3(X,P)-(2)</td></tr> | ||
+ | <tr><td>4</td><td>-</td></tr> | ||
+ | <tr><td>5</td><td>-</td></tr> | ||
+ | <tr><td>6</td><td>10/11 AT-His(X,P)</td></tr> | ||
+ | <tr><td>7</td><td>-</td></tr> | ||
+ | <tr><td>8</td><td>1kb ladder</td></tr> | ||
+ | <tr><td>9</td><td>-</td></tr> | ||
+ | <tr><td>10</td><td>10/11 T7-RBS-His (S,P)-(2)</td></tr> | ||
+ | <tr><td>11</td><td>-</td></tr> | ||
+ | <tr><td>12</td><td>-</td></tr> | ||
+ | <tr><td>13</td><td>10/11 T7-RBS-His (S,P)-(3)</td></tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/thumb/8/81/Img_0003.jpg/776px-Img_0003.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/thumb/1/14/IMG_0003-1-1.jpg/766px-IMG_0003-1-1.jpg"> | ||
+ | |||
+ | <h4>Miniprep</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>1. 10/13 AT (X,P)(Gel Extraction Product)</td><td>43.1</td><td>1.88</td><td>0.77</td></tr> | ||
+ | <tr><td>2. 10/13 AT-His (X,P)(Gel Extraction Product)</td><td>34.6</td><td>1.96</td><td>0.37</td></tr> | ||
+ | <tr><td>3. 10/13 T7-RBS-His(2) (S,P)(Gel Extraction Product)</td><td>57.7</td><td>1.84</td><td>1.29</td></tr> | ||
+ | <tr><td>4. 10/13 T7-RBS-His(3) (S,P)(Gel Extraction Product)</td><td>37.5</td><td>1.81</td><td>1.10</td></tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | <h4>Ligation</h4> | ||
+ | <span class="kyoto-author">Yausda</span> | ||
+ | <table> | ||
+ | <tr><th colspan=2>Vector/(µL)</th><th colspan=2>Insert/(µL)</th><th>Ligation high/(µL)</th><th>Total/(µL)</tr> | ||
+ | |||
+ | <tr><td>1. 9/19 T7-RBS(S,P)(DNA purification product)</td><td>0.8</td><td>10/13 AT(X,P)(Gel Extraction Product)</td><td>2.0</td><td>2.8</td><td>5.6</td></tr> | ||
+ | |||
+ | <tr><td>2. 10/13 T7-RBS-His(S,P)-(2) (Gel Extraction Product)</td><td>0.7</td><td>10/13 AT-His(X,P)(Gel Extraction Product)</td><td>2.5</td><td>3.2</td><td>6.4</td></tr> | ||
+ | |||
+ | <tr><td>3. 10/13 T7-RBS-His(S,P)-(3) (Gel Extraction Product)</td><td>0.8</td><td>10/13 AT-His(X,P)(Gel Extraction Product)</td><td>1.9</td><td>2.7</td><td>5.4</td></tr> | ||
+ | <tr><td>4. 9/19 T7-RBS(S,P)(DNA purification product)</td><td>0.8</td><td>MilliQ</td><td>2.0</td><td>2.8</td><td>5.6</td></tr> | ||
+ | <tr><td>5. 10/13 T7-RBS-His(S,P)-(2) (Gel Extraction Product)</td><td>0.7</td><td>MilliQ</td><td>2.5</td><td>3.2</td><td>6.4</td></tr> | ||
+ | <tr><td>6. 10/13 T7-RBS-His(S,P)-(3) (Gel Extraction Product)</td><td>0.8</td><td>MilliQ</td><td>1.9</td><td>2.7</td><td>5.4</td></tr> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | |||
+ | <h4>Transformation</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr><th colspan=2>Sample/(µL)</th><th colspan=2>CompetentCells/(µL)</th><th>Total/(µL)</th><th>Medium</th></tr> | ||
+ | <tr><td>10/13 Ligation Product (from 1 to 6)</td><td>2</td><td>DH5α</td><td>10</td><td>12</td><td>CP</td></tr> | ||
+ | <tr><td>T7-RBS (1)(Miniprep product)</td><td>2</td><td>BL21(DE3)</td><td>10</td><td>12</td><td>CP</td></tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | ||
+ | <tr><td>10/13 T7-RBS (1)</td><td>20</td><td>-</td><td>-</td><td>3</td><td>3</td><td>B</td><td>12</td><td>1.2</td><td>79.8</td><td>120</td></tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <h4>Cloning<h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>2x KAPA HiFi HotStart ReadyMix/(µL)</th> | ||
+ | <th>Template DNA #1/(µL)</th> | ||
+ | <th>Primers #2/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>12.5</td> | ||
+ | <td>pick up</td> | ||
+ | <td>0.75</td> | ||
+ | <td>11</td> | ||
+ | <td>25</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <br> | ||
+ | <p> | ||
+ | (1)<br> | ||
+ | #1: 10/10 SAMmt-pSB1C3(3) (After mutation, miniprep product)<br> | ||
+ | #2: VF2, Rv SAMmt-His<br> | ||
+ | <br> | ||
+ | (2)<br> | ||
+ | #1: 10/10 SAMmt-pSB1C3(3) (After mutation, miniprep product)<br> | ||
+ | #2: SAMmt(OE-A), Rv SAMmt-His | ||
+ | |||
+ | </p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Last Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>95°C</td> | ||
+ | <td>98°C</td> | ||
+ | <td>60°C</td> | ||
+ | <td>72°C</td> | ||
+ | <td>72°C</td> | ||
+ | <td>12°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5min</td> | ||
+ | <td>20sec</td> | ||
+ | <td>15sec</td> | ||
+ | <td>1min15sec</td> | ||
+ | <td>5min</td> | ||
+ | <td>Infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | |||
+ | |||
+ | <h4>DNA Purification</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>1. 10/14 SAMmt-pSB1C3(3)(PCR product)</td><td>206.7</td><td>1.88</td><td>11.17</td></tr> | ||
+ | <tr><td>2. 10/14 SAMmt-pSB1C3(3)(PCR product)</td><td>187.9</td><td>1.89</td><td>28.58</td></tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | ||
+ | |||
+ | <tr><td>10/11 AT-pSB1C3 (2)(Miniprep product)</td><td>5</td><td>-</td><td>0.5</td><td>-</td><td>0.5</td><td>D</td><td>2</td><td>0.2</td><td>11.8</td><td>20</td></tr> | ||
+ | |||
+ | <tr><td>10/14 SAMmt-His (1)(DNA purification product)</td><td>10</td><td>-</td><td>1</td><td>-</td><td>1</td><td>D</td><td>2</td><td>0.4</td><td>23.6</td><td>40</td></tr> | ||
+ | |||
+ | <tr><td>10/14 SAMmt-His (2)(DNA purification product)</td><td>10</td><td>-</td><td>1</td><td>-</td><td>1</td><td>D</td><td>2</td><td>0.4</td><td>23.6</td><td>40</td></tr> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | <div> | ||
+ | |||
+ | <a name="1014" class="kyoto-jump"></a> | ||
+ | <h3>10/14</h3> | ||
+ | |||
+ | <h4>Gel Extraction</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <p>(1)</p><br> | ||
+ | <table> | ||
+ | <tr><th>Lane</th><th>Sample</th></tr> | ||
+ | <tr><td>1</td><td>1kb ladder</td></tr> | ||
+ | <tr><td>2</td><td>-</td></tr> | ||
+ | <tr><td>3</td><td>10/14 AT(X,P)</td></tr> | ||
+ | <tr><td>4</td><td>-</td></tr> | ||
+ | <tr><td>5</td><td>-</td></tr> | ||
+ | <tr><td>6</td><td>10/14 SAMmt-His(2)(X,P)</td></tr> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <p>(2)</p><br> | ||
+ | <table> | ||
+ | <tr><th>Lane</th><th>Sample</th></tr> | ||
+ | <tr><td>1</td><td>-</td></tr> | ||
+ | <tr><td>2</td><td>-</td></tr> | ||
+ | <tr><td>3</td><td>10/13 T7-RBS(S,P)</td></tr> | ||
+ | <tr><td>4</td><td>1kb ladder</td></tr> | ||
+ | <tr><td>5</td><td>-</td></tr> | ||
+ | <tr><td>6</td><td>10/11 T7-RBS-His(3) (S,P)</td></tr> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2014/thumb/1/17/IMG.jpg/782px-IMG.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/thumb/6/6f/Img-1_0.44.59.jpg/769px-Img-1_0.44.59.jpg"> | ||
+ | |||
+ | <h4>Transformation</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr><th colspan=2>Sample/(µL)</th><th colspan=2>CompetentCells/(µL)</th><th>Total/(µL)</th><th>Medium</th></tr> | ||
+ | |||
+ | <tr><td>10/13 Ligation product (from 1 to 6)</td><td>2</td><td>DH5α</td><td>10</td><td>12</td><td>CP</td></tr> | ||
+ | |||
+ | <tr><td>10/13 T7-RBS(1)</td><td>2</td><td>BL21(DE3)</td><td>10</td><td>12</td><td>CP</td></tr> | ||
+ | |||
+ | <tr><td>Control (pBR322)</td><td>2</td><td>DH5α</td><td>10</td><td>12</td><td>Amp</td></tr> | ||
+ | |||
+ | <tr><td>Control (pBR322)</td><td>2</td><td>DH5α</td><td>10</td><td>12</td><td>Amp</td></tr> | ||
+ | |||
+ | <tr><td>Control (pBR322)</td><td>2</td><td>BL21(DE3)</td><td>10</td><td>12</td><td>Amp</td></tr> | ||
+ | |||
+ | |||
+ | |||
+ | </div> | ||
+ | <div> | ||
+ | |||
+ | <a name="1015" class="kyoto-jump"></a> | ||
+ | <h3>10/15</h3> | ||
+ | |||
+ | |||
+ | <h4>Cloning<h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>2x KAPA HiFi HotStart ReadyMix/(µL)</th> | ||
+ | <th>9/22 T7-RBS-dddD(After mutation, miniprep product)(3)/(µL)</th> | ||
+ | <th>VF2/(µL)</th> | ||
+ | <th>Rv dddD cloning for His/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>12.5</td> | ||
+ | <td>pick up</td> | ||
+ | <td>0.75</td> | ||
+ | <td>0.75</td> | ||
+ | <td>11</td> | ||
+ | <td>25</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Predenature</th> | ||
+ | <th>Denature</th> | ||
+ | <th>Annealing</th> | ||
+ | <th>Extension</th> | ||
+ | <th>Last Extension</th> | ||
+ | <th>Cooling</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>95°C</td> | ||
+ | <td>98°C</td> | ||
+ | <td>60°C</td> | ||
+ | <td>72°C</td> | ||
+ | <td>72°C</td> | ||
+ | <td>12°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5min</td> | ||
+ | <td>20sec</td> | ||
+ | <td>15sec</td> | ||
+ | <td>1min15sec</td> | ||
+ | <td>5min</td> | ||
+ | <td>Infinity</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Denature-Annealing-Extension 30cycle</p> | ||
+ | |||
+ | |||
+ | |||
+ | <h4>DNA Purification</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr><th>Sample</th><th>Concentration/(µg/mL)</th><th>260/280</th><th>260/230</th></tr> | ||
+ | <tr><td>10/14 AT(X,P)(Gel Extraction product)</td><td>32</td><td>1.82</td><td>1.21</td></tr> | ||
+ | <tr><td>10/14 SAMmt-His(2)(X,P)(Gel Extraction product)</td><td>70</td><td>1.78</td><td>1.01</td></tr> | ||
+ | <tr><td>10/14 T7-RBS(S,P)(Gel Extraction product)</td><td>88</td><td>1.66</td><td>2.04</td></tr> | ||
+ | <tr><td>10/14 T7-RBS-His(3) (S,P)(Gel Extraction product)</td><td>156</td><td>1.54</td><td>1.10</td></tr> | ||
+ | <tr><td>10/15 dddD(After mutation, PCR product)</td><td>346</td><td>1.85</td><td>1.44</td></tr> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <h4>Restriction Enzyme Digestion</h4> | ||
+ | <span class="kyoto-author">yasuda</span> | ||
+ | <table> | ||
+ | <tr><th colspan=2>Sample/(µL)</th><th>EcoR1/(µL)</th><th>Xba1/(µL)</th><th>Spe1/(µL)</th><th>Pst1/(µL)</th><th colspan=2>Buffer/(µL)</th><th>BSA/(µL)</th><th>MilliQ/(µL)</th><th>Total/(µL)</th></tr> | ||
+ | |||
+ | <tr><td>10/15 dddD(After mutation, DNA purification product)</td><td>5.8</td><td>-</td><td>1</td><td>-</td><td>1</td><td>D</td><td>4</td><td>0.4</td><td>27.8</td><td>40</td></tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Liquid Culture</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr><th>Sample</th><th>Medium</th></tr> | ||
+ | <tr><td>9/8 T'-RBS-pSB1C3(1)</td><td>CP</td></tr> | ||
+ | <tr><td>REDOX-pSB1C3</td><td>CP</td></tr> | ||
+ | <tr><td>10/3 AT-pSB1C3</td><td>CP</td></tr> | ||
+ | <tr><td>T7-RBS-His(2)</td><td>CP</td></tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | |||
+ | </div> | ||
+ | <div> | ||
+ | |||
+ | <a name="1016" class="kyoto-jump"></a> | ||
+ | <h3>10/16</h3> | ||
+ | |||
+ | <h4>Gel Extraction/Electrophoresis</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Lane</th> | ||
+ | <th>Sample</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>1kbpGeneRuler</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>dddD(His)X,P</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/thumb/3/3e/IMG_0006.jpg/764px-IMG_0006.jpg"> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/thumb/9/91/IMG_0006-1.jpg/747px-IMG_0006-1.jpg"> | ||
+ | |||
+ | <h4>Miniprep</h4> | ||
+ | <span class="kyoto-author">Yasuda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration/(µg/mL)</th> | ||
+ | <th>260/280</th> | ||
+ | <th>260/230</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7RBS-pSB1C3 (1) 10/16 Miniprep product</td> | ||
+ | <td>471</td> | ||
+ | <td>1.90</td> | ||
+ | <td>1.85</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7RBS-pSB1C3 (2) 10/16 Miniprep product A</td> | ||
+ | <td>380</td> | ||
+ | <td>1.92</td> | ||
+ | <td>2.03</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T7RBS-pSB1C3 (2) 10/16 Miniprep product B</td> | ||
+ | <td>372</td> | ||
+ | <td>1.92</td> | ||
+ | <td>1.64</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>AC-pSB1C3(2) 10/16 Miniprep product</td> | ||
+ | <td>473</td> | ||
+ | <td>1.90</td> | ||
+ | <td>1.68</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>REDOX-pSB1C3 10/16 Miniprep product</td> | ||
+ | <td>530</td> | ||
+ | <td>1.93</td> | ||
+ | <td>1.81</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dddD(His)X,P 10/16 gel extraction product</td> | ||
+ | <td>55</td> | ||
+ | <td>1.40</td> | ||
+ | <td>1.36</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>QpSB1C3 10/16 Miniprep product</td> | ||
+ | <td>276</td> | ||
+ | <td>1.86</td> | ||
+ | <td>2.01</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PLQ-4A5 10/16 Miniprep product</td> | ||
+ | <td>370</td> | ||
+ | <td>1.87</td> | ||
+ | <td>2.07</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Ligation</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <p>Toyobo Ligationhigh</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Vector/(µL)</th> | ||
+ | <th colspan=2>Insert/(µL)</th> | ||
+ | <th colspan=2>Ligase/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</tr> | ||
+ | <tr> | ||
+ | <td>10/14 T7-RBS S,P</td> | ||
+ | <td>2</td> | ||
+ | <td>10/14 AT X,P</td> | ||
+ | <td>2</td> | ||
+ | <td>2xligationhigh</td> | ||
+ | <td>4</td> | ||
+ | <td>-</td> | ||
+ | <td>8</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/14 T7-RBS S,P</td> | ||
+ | <td>2</td> | ||
+ | <td>10/14 AT X,P</td> | ||
+ | <td>-</td> | ||
+ | <td>2xligationhigh</td> | ||
+ | <td>4</td> | ||
+ | <td>2</td> | ||
+ | <td>8</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/14 T7-RBS-His S,P</td> | ||
+ | <td>1</td> | ||
+ | <td>10/13 AT-His X,P</td> | ||
+ | <td>2</td> | ||
+ | <td>2xligationhigh</td> | ||
+ | <td>4</td> | ||
+ | <td>1</td> | ||
+ | <td>8</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/14 T7-RBS-His S,P</td> | ||
+ | <td>1</td> | ||
+ | <td>10/13 AT-His X,P</td> | ||
+ | <td>-</td> | ||
+ | <td>2xligationhigh</td> | ||
+ | <td>4</td> | ||
+ | <td>3</td> | ||
+ | <td>8</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Promega</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Vector/(µL)</th> | ||
+ | <th colspan=2>Insert/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th colspan=2>Ligase/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</tr> | ||
+ | <tr> | ||
+ | <td>10/14 T7-RBS S,P</td> | ||
+ | <td>2</td> | ||
+ | <td>10/14 AT X,P</td> | ||
+ | <td>2</td> | ||
+ | <td>2xRapidLigationBuffer</td> | ||
+ | <td>5</td> | ||
+ | <td>T4Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/14 T7-RBS S,P</td> | ||
+ | <td>2</td> | ||
+ | <td>10/14 AT X,P</td> | ||
+ | <td>-</td> | ||
+ | <td>2xRapidLigationBuffer</td> | ||
+ | <td>5</td> | ||
+ | <td>T4Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>2</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/14 T7-RBS-His S,P</td> | ||
+ | <td>1</td> | ||
+ | <td>10/13 AT-His X,P</td> | ||
+ | <td>2</td> | ||
+ | <td>2xRapidLigationBuffer</td> | ||
+ | <td>5</td> | ||
+ | <td>T4Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>1</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/14 T7-RBS-His S,P</td> | ||
+ | <td>2</td> | ||
+ | <td>10/14 SAM-His X,P</td> | ||
+ | <td>2</td> | ||
+ | <td>2xRapidLigationBuffer</td> | ||
+ | <td>5</td> | ||
+ | <td>T4Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/14 T7-RBS-His S,P</td> | ||
+ | <td>2</td> | ||
+ | <td>REDOX-His</td> | ||
+ | <td>2</td> | ||
+ | <td>2xRapidLigationBuffer</td> | ||
+ | <td>5</td> | ||
+ | <td>T4Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>-</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/14 T7-RBS-His S,P</td> | ||
+ | <td>2</td> | ||
+ | <td>REDOX-His</td> | ||
+ | <td>-</td> | ||
+ | <td>2xRapidLigationBuffer</td> | ||
+ | <td>5</td> | ||
+ | <td>T4Ligase</td> | ||
+ | <td>1</td> | ||
+ | <td>2</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>NewIngland BioLabs</p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Vector/(µL)</th> | ||
+ | <th colspan=2>Insert/(µL)</th> | ||
+ | <th colspan=2>Buffer/(µL)</th> | ||
+ | <th colspan=2>Ligase/(µL)</th> | ||
+ | <th>MilliQ/(µL)</th> | ||
+ | <th>Total/(µL)</tr> | ||
+ | <tr> | ||
+ | <td>10/14 T7-RBS-His S,P</td> | ||
+ | <td>2</td> | ||
+ | <td>10/14 SAM-His X,P</td> | ||
+ | <td>2</td> | ||
+ | <td>10xLigationBuffer</td> | ||
+ | <td>0.5</td> | ||
+ | <td>T4 Ligase</td> | ||
+ | <td>0.5</td> | ||
+ | <td>-</td> | ||
+ | <td>5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/14 T7-RBS-His S,P</td> | ||
+ | <td>2</td> | ||
+ | <td>REDOX-His</td> | ||
+ | <td>2</td> | ||
+ | <td>10xLigationBuffer</td> | ||
+ | <td>0.5</td> | ||
+ | <td>T4 Ligase</td> | ||
+ | <td>0.5</td> | ||
+ | <td>-</td> | ||
+ | <td>5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/14 T7-RBS-His S,P</td> | ||
+ | <td>2</td> | ||
+ | <td>REDOX-His</td> | ||
+ | <td>-</td> | ||
+ | <td>10xLigationBuffer</td> | ||
+ | <td>0.5</td> | ||
+ | <td>T4 Ligase</td> | ||
+ | <td>0.5</td> | ||
+ | <td>2</td> | ||
+ | <td>5</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h4>Transformation</h4> | ||
+ | <span class="kyoto-author">Honda</span> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th colspan=2>Sample/(µL)</th> | ||
+ | <th colspan=2>CompetentCells/(µL)</th> | ||
+ | <th>Total/(µL)</th> | ||
+ | <th>Medium</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Toyobo T7-AT</td> | ||
+ | <td>2</td> | ||
+ | <td>BL21</td> | ||
+ | <td>10</td> | ||
+ | <td>12</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Toyobo T7-AT control</td> | ||
+ | <td>2</td> | ||
+ | <td>BL21</td> | ||
+ | <td>10</td> | ||
+ | <td>12</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Toyobo T7-AT-His</td> | ||
+ | <td>2</td> | ||
+ | <td>BL21</td> | ||
+ | <td>10</td> | ||
+ | <td>12</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Toyobo T7-AT-His control</td> | ||
+ | <td>2</td> | ||
+ | <td>BL21</td> | ||
+ | <td>10</td> | ||
+ | <td>12</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10/13 Miniprep product T7-RBS(1)B</td> | ||
+ | <td>9</td> | ||
+ | <td>BL21</td> | ||
+ | <td>90</td> | ||
+ | <td>99</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Toyobo T7-AT</td> | ||
+ | <td>4</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>20</td> | ||
+ | <td>24</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Toyobo T7-AT control</td> | ||
+ | <td>4</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>20</td> | ||
+ | <td>24</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Toyobo T7-AT-His</td> | ||
+ | <td>4</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>20</td> | ||
+ | <td>24</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Toyobo T7-AT-His control</td> | ||
+ | <td>4</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>20</td> | ||
+ | <td>24</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Promega T7-AT</td> | ||
+ | <td>5</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>30</td> | ||
+ | <td>35</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Promega T7-AT control</td> | ||
+ | <td>5</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>30</td> | ||
+ | <td>35</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Promega T7-AT-His</td> | ||
+ | <td>5</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>30</td> | ||
+ | <td>35</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Promega T7-SAM-His</td> | ||
+ | <td>5</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>30</td> | ||
+ | <td>35</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Promega T7-REDOX-His</td> | ||
+ | <td>5</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>30</td> | ||
+ | <td>35</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Promega T7-REDOX-His control</td> | ||
+ | <td>5</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>30</td> | ||
+ | <td>35</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>NewIngland T7-SAM-His</td> | ||
+ | <td>5</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>30</td> | ||
+ | <td>35</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>NewIngland T7-REDOX-His</td> | ||
+ | <td>5</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>30</td> | ||
+ | <td>35</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>NewIngland T7-REDOX-His control</td> | ||
+ | <td>5</td> | ||
+ | <td>DH5α</td> | ||
+ | <td>30</td> | ||
+ | <td>35</td> | ||
+ | <td>CP</td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
<!-- 警告:ここより下を消したら怒る --> | <!-- 警告:ここより下を消したら怒る --> | ||
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Latest revision as of 03:46, 18 October 2014
7/31
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|
3/24 dddD PCR product | 3.2 | 1 | - | 1 | - | H | 3 | - | 21.8 | 30 |
pSB1C3 | 18.5 | 1 | - | 1 | - | H | 3 | - | 6.5 | 30 |
pSB1C3 control | 6.2 | - | - | - | - | H | 1 | - | 2.8 | 10 |
8/1
Gel Extraction
Lane | Sample |
---|---|
1 | 1kbp ladder |
2 | 3/24 dddD PCR product E,S |
3 | 3/24 dddD PCR product E,S |
4 | Blank |
5 | pSB1C3 E,S |
6 | pSB1C3 E,S |
7 | Blank |
8 | 1kbp ladder |
9 | 3/24 dddD PCR product control |
10 | pSB1C3 control |
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
pSB1C3 | 13.3 | 1.90 | - |
dddD | 10.8 | 1.64 | 0.15 |
Ligation
Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) | ||||
---|---|---|---|---|---|---|---|---|---|
8/1 pSB1C3 gel extraction product 2047bp | 1.5 | 8/1 dddD gel extraction product 2545bp | 6.9 | 10xT4Ligase | 2 | T4Ligase | 1 | 8.6 | 20 |
Transformation
Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium | ||
---|---|---|---|---|---|
pSB1C3-dddD | 3 | Self-Making Competent Cells (DH5α) | 30 | 33 | SOC |
8/2
PCR Cloning
dddD without Pre-Suf | 2x KAPA HiFi Hotstart Ready Mix/(µL) | F Pre-dddD PCR product 62/(µL) | Rv Spe1-dddD PCR product 68/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
pick up | 12.5 | 0.75 | 0.75 | 11 | 25 |
Predenature | Denature | Annealing | Extension |
---|---|---|---|
95°C | 98°C | 65°C | 72°C |
5min | 20sec | 15sec | 50sec |
Denature-Annealing-Extension 25cycle
DNA Purification
8/2 Pre-dddD-Suf PCR product
Colony PCR
Master Mix
Quick Taq/(µL) | VF2/(µL) | VR/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
15 | 0.6 | 0.6 | 13.8 | 30 |
Predenature | Denature | Annealing | Extension |
---|---|---|---|
94°C | 94°C | 55°C | 68°C |
2min | 30sec | 30sec | 24sec |
Denature-Annealing-Extension 30cycle
Number | Sample |
---|---|
1 | pSB1C3-dddD(1) |
2 | pSB1C3-dddD(2) |
3 | pSB1C3-dddD(3) |
Concentration Measurement
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
dddD PCR product | 245 | 1.59 | 2.17 |
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|
8/2 dddD PCR product | 8.2 | 1 | - | 1 | - | H | 3 | - | 16.8 | 30 |
8/2 dddD PCR product control | 0.41 | - | - | - | - | H | 1 | - | 8.6 | 10 |
pSB1C3 | 12.7 | 1 | - | 1 | - | H | 3 | - | 12.3 | 30 |
pSB1C3 control | 0.04 | - | - | - | - | H | 1 | - | 8.4 | 10 |
8/3
Electrophoresis
Lane | Sample |
---|---|
1 | pSB1C3-dddD(1) |
2 | pSB1C3-dddD(2) |
3 | pSB1C3-dddD(3) |
4 | 1kb ladder (3µL) |
5 | 1kb ladder (1µL) |
Gel Extraction
Lane | Sample |
---|---|
1 | 1kb ladder |
2 | Blank |
3 | pSB1C3 2070bp |
4 | Blank |
5 | pSB1C3 |
6 | Blank |
7 | 8/2 dddD PCR product 2544bp |
8 | Blank |
9 | 8/2 dddD PCR product |
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
pSB1C3 | 12.5 | 1.37 | 0.42 |
dddD PCR product | 35 | 1.54 | 0.85 |
Ligation
Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) | ||||
---|---|---|---|---|---|---|---|---|---|
pSB1C3 | 7 | dddD | 7 | 2x Buffer | 15 | T4 Ligase | 1 | - | 30 |
pSB1C3 | 7 | dddD | 14 | 2x Buffer | 22 | T4 Ligase | 1 | - | 44 |
pSB1C3 | 7 | dddD | - | 2x Buffer | 15 | T4 Ligase | 1 | 7 | 30 |
Transformation
Sample | CompetentCells | Medium |
---|---|---|
(1) | Self-Making Competent Cells (DH5α) | CP |
(2) | Self-Making Competent Cells (DH5α) | CP |
8/4
Colony PCR
Master Mix
Quick Taq/(µL) | VF2/(µL) | VR/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
40 | 1.6 | 1.6 | 36.8 | 80 |
Predenature | Denature | Annealing | Extension |
---|---|---|---|
94°C | 94°C | 55°C | 68°C |
2min | 30sec | 30sec | 24sec |
Denature-Annealing-Extension 30cycle
Lane | Sample |
---|---|
1 | dddD+pSB1C3(1)-1 |
2 | dddD+pSB1C3(1)-2 |
3 | dddD+pSB1C3(2)-1 |
4 | dddD+pSB1C3(2)-2 |
5 | dddD+pSB1C3(2)-3 |
6 | dddD+pSB1C3(2)-4 |
7 | pSB1C3 control-1 |
8 | control |
Electrophoresis
Lane | Sample |
---|---|
1 | 1kbp ladder |
2 | dddD+pSB1C3(1)-1 |
3 | dddD+pSB1C3(1)-2 |
4 | dddD+pSB1C3(2)-1 |
5 | dddD+pSB1C3(2)-2 |
6 | dddD+pSB1C3(2)-3 |
7 | dddD+pSB1C3(2)-4 |
8 | pSB1C3 control-1 |
9 | control |
10 | 1kbp ladder |
PCR
Unexpected bands are confirmed in former Electrophoresis, so contamination of MilliQ or Quicktaq is suspected. For confirming contamination, PCR and Electrophoresis are conducted.
* suspicious samples for contamination
(1)
*Quick Taq/(µL) | VF2/(µL) | VR/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
5 | 0.2 | 0.2 | 4.6 | 10 |
(2)
Quick Taq/(µL) | VF2/(µL) | VR/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
5 | 0.2 | 0.2 | 4.6 | 10 |
(3)
Quick Taq/(µL) | VF2/(µL) | VR/(µL) | *MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
5 | 0.2 | 0.2 | 4.6 | 10 |
(4)
*Quick Taq/(µL) | VF2/(µL) | VR/(µL) | *MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
5 | 0.2 | 0.2 | 4.6 | 10 |
Predenature | Denature | Annealing | Extension |
---|---|---|---|
94°C | 94°C | 55°C | 68°C |
2min | 30sec | 30sec | 24sec |
Denature-Annealing-Extension 30cycle
Electrophoresis
Lane | Sample |
---|---|
1 | 1kbp ladder |
2 | (1) |
3 | (2) |
4 | (3) |
5 | (4) |
8/18
PCR
For confirming contamination of VF2 and VR primers, we pour samples into new tubes and perform PCR and Electrophoresis.
(1)
Quick Taq/(µL) | VF2/(µL) | VR/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
5 | 0.2 | 0.2 | 4.6 | 10 |
control(2)
Quick Taq/(µL) | VF2/(µL) | VR/(µL) | 8/2 Pcon3/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
5 | 0.2 | 0.2 | 0.6 | 4.6 | 10 |
(3)
Quick Taq/(µL) | VF2/(µL) | VR/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
5 | 0.2 | 0.2 | 4.6 | 10 |
(4)
Quick Taq/(µL) | VF2/(µL) | VR/(µL) | 8/2 Pcon3/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
5 | 0.2 | 0.2 | 0.6 | 4.0 | 10 |
Predenature | Denature | Annealing | Extension |
---|---|---|---|
94°C | 94°C | 55°C | 68°C |
2min | 30sec | 30sec | 24sec |
Denature-Annealing-Extension 30cycle
Electrophoresis
Number | Sample |
---|---|
1 | 1kbp ladder |
2 | (1) |
3 | (2) |
4 | (3) |
5 | (4) |
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|
dddD 8/2 PCR product | 5.5 | 0.7 | - | 0.7 | - | H | 2 | - | 11.1 | 20 |
dddD 8/2 PCR product control | 0.41 | - | - | - | - | H | 1 | - | 8.6 | 10 |
pSB1C3 | 12.7 | 1 | - | 1 | - | H | 3 | - | 8.4 | 10 |
pSB1C3 control | 0.64 | - | - | - | - | H | 1 | - | 8.4 | 10 |
Colony PCR
Master Mix
Quick Taq/(µL) | VF2/(µL) | VR/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
40 | 1.6 | 1.6 | 36.8 | 80 |
Predenature | Denature | Annealing | Extension |
---|---|---|---|
94°C | 94°C | 55°C | 68°C |
2min | 30sec | 30sec | 24sec |
Denature-Annealing-Extension 30cycle
Number | Sample |
---|---|
1 | dddD+pSB1C3(1)-1 |
2 | dddD+pSB1C3(1)-2 |
3 | dddD+pSB1C3(2)-1 |
4 | dddD+pSB1C3(2)-2 |
5 | dddD+pSB1C3(2)-3 |
6 | dddD+pSB1C3(2)-4 |
7 | pSB1C3 control-1 |
8 | control |
Electrophoresis
Lane | Sample |
---|---|
1 | 1kbp ladder |
2 | dddD+pSB1C3(1)-1 |
3 | dddD+pSB1C3(1)-2 |
4 | dddD+pSB1C3(2)-1 |
5 | dddD+pSB1C3(2)-2 |
6 | dddD+pSB1C3(2)-3 |
7 | dddD+pSB1C3(2)-4 |
8 | pSB1C3 control-1 |
9 | control |
Gel Extraction
Lane | Sample |
---|---|
1 | 1kbp ladder |
2 | dddD control |
3 | dddD E,S |
4 | Blank |
5 | pSB1C3 control |
6 | pSB1C3 E,S |
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
8/18 pSB1C3 | 105 | 1.30 | 0.85 |
8/18 dddD | 105 | 1.27 | 0.73 |
8/19
Ligation
(1)
Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) | ||||
---|---|---|---|---|---|---|---|---|---|
pSB1C3 | 3.6 | dddD | 1 | 2xBuffer | 5.6 | T4Ligase | 1 | - | 11.2 |
(2)
Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) | ||||
---|---|---|---|---|---|---|---|---|---|
pSB1C3 | 3.6 | - | - | 2xBuffer | 5.6 | T4Ligase | 1 | 1 | 11.2 |
8/20
Transformation
Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium | ||
---|---|---|---|---|---|
8/19 Ligation (1) | 11.2 | Competent Cells (DH5α) | 50 | 61.2 | SOC |
8/19 Ligation (2) | 11.2 | Competent Cells (DH5α) | 50 | 61.2 | SOC |
Ligation
(1)
Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) | ||||
---|---|---|---|---|---|---|---|---|---|
pSB1C3 | 10.8 | dddD | 3 | 2xBuffer | 16.8 | T4Ligase | 3 | - | 33.6 |
(2)
Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) | ||||
---|---|---|---|---|---|---|---|---|---|
pSB1C3 control | 10.8 | - | - | 2xBuffer | 16.8 | T4Ligase | 3 | 3 | 33.6 |
Transformation
Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium | ||
---|---|---|---|---|---|
dddD+pSB1C3 | 5 | Competent Cells (DH5α) | 20 | 25 | SOC |
control | 5 | Competent Cells (DH5α) | 20 | 25 | SOC |
8/21
Colony PCR
Master Mix
Quick Taq/(µL) | VF2/(µL) | VR/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
35 | 1.4 | 1.4 | 32.2 | 70 |
Predenature | Denature | Annealing | Extension |
---|---|---|---|
94°C | 94°C | 55°C | 68°C |
2min | 30sec | 30sec | 24sec |
Denature-Annealing-Extension 30cycle
Transformation
Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium | ||
---|---|---|---|---|---|
AT-His-YCplac111 | 10 | DH5α | 2 | 12 | CP |
REDOX-YCplac111 | 10 | DH5α | 2 | 12 | CP |
DECARB-pSB1C3 | 10 | DH5α | 2 | 12 | CP |
DiDECARB-pSB1C3 | 10 | DH5α | 2 | 12 | CP |
Ligation
Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) | ||||
---|---|---|---|---|---|---|---|---|---|
9/15 T7-RBS(S,P)(Dephosphorylation product) | 2.0 | 9/15 dddD(After mutation)(X,P)(Gel extraction product) | 2.0 | (x2)Buffer | 5 | T4 Ligase | 1 | 0 | 10 |
9/15 pSB1C3 (E,P)(Dephosphorylation product) | 0.4 | 9/12 SAMmt (E,P)(DNA purification product) | 2.9 | (x2)Buffer | 5 | T4 Ligase | 1 | 0.7 | 10 |
9/15 pSB1C3 (E,P)(Dephosphorylation product) | 1.3 | 9/15 REDOX (E,P)(DNA purification product) | 2.4 | (x2)Buffer | 5 | T4 Ligase | 1 | 0.3 | 10 |
9/15 T7-RBS(S,P)(Gel extraction product) | 2.6 | 9/15 AT His (X,P)(DNA purification product) | 1.2 | (x2)Buffer | 5 | T4 Ligase | 1 | 0.2 | 10 |
Transformation
Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium | ||
---|---|---|---|---|---|
DECARB-YCplac111 | 2 | DH5α | 10 | 12 | SOD |
DiDECARB-YCplac111 | 2 | DH5α | 10 | 12 | SOD |
dddD(After mutation)-T7-RBS-pSB1C3 | 2 | DH5α | 10 | 12 | SOD |
SAMmt-pSB1C3 | 2 | DH5α | 10 | 12 | SOD |
REDOX-pSB1C3 | 2 | DH5α | 10 | 12 | SOD |
AT-T7-RBS-pSB1C3 | 2 | DH5α | 10 | 12 | SOD |
DNA Purification
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
9/15 REDOX (E,P) | 45 | - | - |
9/16
Miniprep
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
9/15 AT-pSB1C3-(1) | 280 | 1.81 | 2.08 |
9/15 AT-pSB1C3-(1) | 205 | 1.64 | 1.32 |
9/15 AT-pSB1C3-(1) | 320 | 1.66 | 1.61 |
PCR (for Sequence)
9/14 dddD-pSB1C3(Miniprep product)-(1)
(1)dddD sequence VF2
Templete DNA/(µL) | Big Dye/(µL) | (x5)Buffer/(µL) | Primer | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
0.63 | 0.5 | 1.75 | 0.5 | 6.6 | 10.5 |
(2)dddD sequence No.1
Templete DNA/(µL) | Big Dye/(µL) | (x5)Buffer/(µL) | Primer | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
0.63 | 0.5 | 1.75 | 0.5 | 6.6 | 10.5 |
(3)dddD sequence No.2
Templete DNA/(µL) | Big Dye/(µL) | (x5)Buffer/(µL) | Primer | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
0.63 | 0.5 | 1.75 | 0.5 | 6.6 | 10.5 |
(4)dddD sequence No.3
Templete DNA/(µL) | Big Dye/(µL) | (x5)Buffer/(µL) | Primer | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
0.63 | 0.5 | 1.75 | 0.5 | 6.6 | 10.5 |
(5)dddD sequence No.4
Templete DNA/(µL) | Big Dye/(µL) | (x5)Buffer/(µL) | Primer | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
0.63 | 0.5 | 1.75 | 0.5 | 6.6 | 10.5 |
(6)dddD sequence No.5
Templete DNA/(µL) | Big Dye/(µL) | (x5)Buffer/(µL) | Primer | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
0.63 | 0.5 | 1.75 | 0.5 | 6.6 | 10.5 |
(7)dddD sequence No.6
Templete DNA/(µL) | Big Dye/(µL) | (x5)Buffer/(µL) | Primer | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
0.63 | 0.5 | 1.75 | 0.5 | 6.6 | 10.5 |
Predenature | Denature | Annealing | Extension | Final Extension | Cooling |
---|---|---|---|---|---|
96°C | 96°C | 50°C | 60°C | 4°C | 4°C |
2min | 10sec | 5sec | 4min | 1min | Infinity |
Denature-Annealing-Extension 30cycle
9/14 dddD-pSB1C3(Miniprep product)-(2)
(1)dddD sequence VF2
Templete DNA/(µL) | Big Dye/(µL) | (x5)Buffer/(µL) | Primer | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
0.63 | 0.5 | 1.75 | 0.5 | 6.6 | 10.5 |
(2)dddD sequence No.1
Templete DNA/(µL) | Big Dye/(µL) | (x5)Buffer/(µL) | Primer | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
0.63 | 0.5 | 1.75 | 0.5 | 6.6 | 10.5 |
(3)dddD sequence No.2
Templete DNA/(µL) | Big Dye/(µL) | (x5)Buffer/(µL) | Primer | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
0.63 | 0.5 | 1.75 | 0.5 | 6.6 | 10.5 |
(4)dddD sequence No.3
Templete DNA/(µL) | Big Dye/(µL) | (x5)Buffer/(µL) | Primer | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
0.63 | 0.5 | 1.75 | 0.5 | 6.6 | 10.5 |
(5)dddD sequence No.4
Templete DNA/(µL) | Big Dye/(µL) | (x5)Buffer/(µL) | Primer | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
0.63 | 0.5 | 1.75 | 0.5 | 6.6 | 10.5 |
(6)dddD sequence No.5
Templete DNA/(µL) | Big Dye/(µL) | (x5)Buffer/(µL) | Primer | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
0.63 | 0.5 | 1.75 | 0.5 | 6.6 | 10.5 |
(7)dddD sequence No.6
Templete DNA/(µL) | Big Dye/(µL) | (x5)Buffer/(µL) | Primer | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
0.63 | 0.5 | 1.75 | 0.5 | 6.6 | 10.5 |
9/15 AT-pSB1C3(Miniprep product)-(1)
(1)VF
Templete DNA/(µL) | Big Dye/(µL) | (x5)Buffer/(µL) | Primer | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
1.43 | 0.5 | 1.75 | 0.5 | 5.8 | 10.5 |
(2)AT for Sequence
Templete DNA/(µL) | Big Dye/(µL) | (x5)Buffer/(µL) | Primer | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
1.43 | 0.5 | 1.75 | 0.5 | 5.8 | 10.5 |
Predenature | Denature | Annealing | Extension | Final Extension | Cooling |
---|---|---|---|---|---|
96°C | 96°C | 50°C | 60°C | 4°C | 4°C |
2min | 10sec | 5sec | 4min | 1min | Infinity |
Denature-Annealing-Extension 30cycle
Dephosphorylation
Sample/(µL) | |
---|---|
1.*(written in magnetosome) | * |
2.* | * |
3.9/15 T7-RBS-pSB1C3(P) | 30 |
4.9/15 T7-RBS-pSB1C3(S,P) | 30 |
5.9/15 T7-RBS-pSB1C3(S,P) | 30 |
6.9/15 T7-RBS-pSB1C3(S,P) | 30 |
7.9/15 T7-RBS-pSB1C3(S,P) | 30 |
Colony PCR
YasudaMaster Mix
Quick Taq/(µL) | M13 Ew/(µL) | M13 Rv/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
300 | 12 | 12 | 276 | 600 |
Electrophoresis
(1)
Lane | Sample | ||
---|---|---|---|
1-8 | 9/15 REDOX-YCplac111 | ||
9 | 1kb ladder | ||
10-17 | 9/15 DECARB-YCplac111 |
(2)
Lane | Sample | ||
---|---|---|---|
1-8 | 9/15 DiDECARB-YCplac111 | ||
9 | 1kb ladder | ||
10-17 | 9/15 AT-His-YCplac111 |
(3)
Lane | Sample | ||
---|---|---|---|
1 | 1kb ladder | ||
2-8 | 9/15 AT-His-YCplac111 |
(4)
Lane | Sample | ||
---|---|---|---|
1 | 1kb ladder | ||
2-6 | 9/15 AT-His-YCplac111 |
Gel Extraction
(1)
Lane | Sample |
---|---|
1 | 1kb ladder |
2 | - |
3 | (written in "magnetosome") |
4 | - |
5 | 1kb ladder |
6 | - |
7 | (written in "magnetosome") |
8 | - |
9 | 1kb ladder |
10 | - |
11 | 9/15 pSB1C3(E,P) |
(2)
Lane | Sample |
---|---|
1 | 1kb ladder |
2 | - |
3 | 9/15 T7-RBS-pSB1C3(P) |
4 | - |
5 | 1kb ladder |
6 | - |
7 | 9/15 T7-RBS-pSB1C3(S,P) |
8 | - |
9 | 1kb ladder |
10 | - |
11 | 9/15 T7-RBS-pSB1C3(S,P) |
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
7. 9/15 T7-RBS-pSB1C3(S,P) | 90 | 1.40 | 0.44 |
(3)
Lane | Sample |
---|---|
1 | - |
2 | 1kb ladder |
3 | - |
4 | 9/15 T7-RBS-pSB1C3(S,P) |
5 | - |
6 | 1kb ladder |
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
4. 9/15 T7-RBS-pSB1C3(S,P) | 80 | 1.30 | 1.02 |
DNA Purification
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
9/15 T7-RBS-pSB1C3(S,P)(Dephosphorylation product) | 0.7(x50) | 1.81 | 1.13 |
9/15 pSB1C3(E,P)(Dephosphorylation product) | 1.1(x50) | 1.59 | 1.25 |
Ligation
Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) | ||||
---|---|---|---|---|---|---|---|---|---|
9/16 T7-RBS(S,P)(DNA purification product) | 1.7 | 9/15 dddD(After mutation)(X,P)(gel extraction product) | 2 | (x2)Buffer | 5 | T4 Ligase | 1 | 0.3 | 10 |
9/16 pSB1C3 (E,P)(DNA purification product) | 0.55 | 9/15 SAMmt (E,P)(gel extraction product) | 2.9 | (x2)Buffer | 5 | T4 Ligase | 1 | 0.55 | 10 |
9/16 pSB1C3 (E,P)(DNA purification product) | 1.6 | 9/15 REDOX (E,P)(gel extraction product) | 2.2 | (x2)Buffer | 5 | T4 Ligase | 1 | 0.2 | 10 |
9/16 T7-RBS-His(S,P)(DNA purification product) | 2.6 | 9/15 AT His(X,P)(gel extraction product) | 1.2 | (x2)Buffer | 5 | T4 Ligase | 1 | 0.2 | 10 |
Dephosphorylation
Sample/(µL) | CIAP | Total | |
---|---|---|---|
9/13 pSB1C3 (X,S)(gel extraction product) | 16 | 0.2 | 16.2 |
Transformation
Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium | ||
---|---|---|---|---|---|
9/16 AT-His-T7-RBS-His | - | DH5α | - | - | - |
9/16 REDOX-pSB1C3 | - | DH5α | - | - | - |
9/16 SAMmt-pSB1C3 | - | DH5α | - | - | - |
9/16 dddD-T7-RBS(After mutation) | - | DH5α | - | - | - |
9/16 AT-His-YCplac111 | - | DH5α | - | - | - |
9/16 REDOX-YCplac111 | - | DH5α | - | - | - |
9/16 DECARB-YCplac111 | - | DH5α | - | - | - |
9/16 DiDECARB-YCplac111 | - | DH5α | - | - | - |
DNA Purification
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
9/16 pSB1C3(X,S)(Dephosphorylation product) | 35 | 1.69 | 0.56 |
Liquid Culture
Sample | Medium |
---|---|
9/16 AT RBS His-T7 RBS His(1) | - |
9/16 AT RBS His-T7 RBS His(2) | - |
PCR
(1)
9/11 OE-PCR SAMmt 2nd-(2) | 2x KAPA HiFi Hotstart Ready Mix/(µL) | Fw Primer/(µL) | Rv Primer/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
pick up | 12.5 | 0.75 | 0.75 | 11 | 25 |
9/11 OE-PCR DECARB 2nd-(3) | 2x KAPA HiFi Hotstart Ready Mix/(µL) | Fw Primer/(µL) | Rv Primer/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
pick up | 12.5 | 0.75 | 0.75 | 11 | 25 |
Predenature | Denature | Annealing | Extension |
---|---|---|---|
95°C | 98°C | 65°C | 72°C |
5min | 20sec | 15sec | 50sec |
Denature-Annealing-Extension 25cycle
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|
9/11 OE-PCR SAMmt 2nd-(2) | 8.2 | 0.5 | - | - | 0.5 | H | 1.5 | 0 | 4.3 | 15 |
9/15 AT-pSB1C3-(3)(Miniprep product) | 9.8 | - | 1 | - | 1 | M | 3 | 3 | 12.2 | 30 |
Control | 0.5 | - | - | - | - | M | 1 | 1 | 7.5 | 10 |
Ligation
Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) | ||||
---|---|---|---|---|---|---|---|---|---|
pSB1C3 | 1.1 | 9/12 DECARB(X,E)(DNA purification product) | 2.9 | (x2)Buffer | 5 | T4 Ligase | 1 | 0 | 10 |
pSB1C3 | 0.85 | 9/12 DiDECARB(X,E)(DNA purification product) | 2.2 | (x2)Buffer | 5 | T4 Ligase | 1 | 0.95 | 10 |
Transformation
Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium | ||
---|---|---|---|---|---|
9/16 DECARB-pSB1C3 | 1 | DH5α | 10 | 11 | - |
9/16 DiDECARB-pSB1C3 | 1 | DH5α | 10 | 11 | - |
15min on ice - 1min heat shock(42°C) - 45min preculture (42°C)
9/17
Cycle PCR
16 Cycle
BigDye Terminator v3.1 Cycle Sequencing RR-24/(µL) | BigDye Terminator v3.1 v1.1 5x Sequencing Buffer/(µL) | Primer #1/(pmol) | Templete DNA #2/(ng) | MilliQ /(µL) | Total/(µL) |
---|---|---|---|---|---|
2 | 1 | 1.6 | 75-150 | up to 10 | 10 |
(1)
#1: VF2
#2: 9/11 dddD (miniprep product) 1µL
#1: Sequence No.5
#2: 9/11 dddD (miniprep product) 1µL
(2)
#1: VF2
#2: 9/14 dddD(After mutation)(miniprep product) 0.25µL
#1: VR
#2: 9/14 dddD(After mutation)(miniprep product) 0.25µL
(3)
#1: VF2
#2: 9/14 T7-RBS-His-pSB1C3 (miniprep product) 0.34µL
#1: Sequence No.5
#2: 9/14 T7-RBS-His-pSB1C3 (miniprep product) 0.34µL
(4)
#1: VR
#2: 9/16 AT-pSB1C3-(1) (miniprep product) 0.5µL
#1: VF2
#2: 9/16 AT-pSB1C3-(1) (miniprep product) 0.5µL
#1: Sequence No.1
#2: 9/16 AT-pSB1C3-(1) (miniprep product) 0.5µL
#1: Secuence No.2
#2: 9/16 AT-pSB1C3-(1) (miniprep product) 0.5µL
Predenature | Denature | Annealing | Extension | Final Extension | Cooling |
---|---|---|---|---|---|
96°C | 96°C | 50°C | 60°C | 4°C | 4°C |
2min | 10sec | 5sec | 4min | 1min | Infinity |
Denature-Annealing-Extension 30cycle
Gel Extraction
Lane | Sample |
---|---|
1 | 1kb ladder |
2 | Control |
3 | - |
4 | 9/16 AT-pSB1C3(X,P) |
5 | - |
6 | - |
DNA Purification
Sample | Concentration/(µg/mL) |
---|---|
9/12 SAMmt (E,P) | 75 |
9/16 SAMmt (PCR product) | 140 |
9/16 DECARB (PCR product) | 230 |
PCR
10x Buffer for KOD-Plus-Ver.2 | 2mM dNTPs | 25mM MgSO4 | Primer(10µM each) #1 | Template DNA #2 | KOD-Plus-(1U/µL) | MilliQ | Total |
---|---|---|---|---|---|---|---|
5µL | 5µL | 3µL | 2µL | 0.5µL | 1µL | 31.5µL | 50µL |
(1)
#1: Pre-REDOX, Suf-REDOX
#2: 9/11 REDOX (DNA purification product)
(2)
#1: Pre-REDOX, Rv-REDOX cloning for His
#2: 9/11 REDOX (DNA purification product)
(3)
#1: DiDECARB OE-A, DiDECARB OE-D
#2: 9/11 DiDECARB (for OE-PCR 2nd)(9/12 DNA purification product)
Predenature | Denature | Annealing | Extension | Final Extension | Cooling |
---|---|---|---|---|---|
98°C | 98°C | 55°C | 72°C | 72°C | 12°C |
2min | 10sec | 10sec | 1min | 5min | Infinity |
Denature-Annealing-Extension 25cycle
DNA Purification
Sample | Concentration/(µg/mL) |
---|---|
9/17 REDOX PCR | 435 |
9/17 REDOX-His PCR | 365 |
9/17 DiDECARB PCR | 230 |
Colony PCR
Master Mix
Quick Taq/(µL) | VF2/(µL) | VR/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
85 | 3.4 | 3.4 | 78.2 | 170 |
Predenature | Denature | Annealing | Extension |
---|---|---|---|
94°C | 94°C | 55°C | 68°C |
2min | 30sec | 30sec | 2min30sec |
Denature-Annealing-Extension 30cycle
Number | Sample |
---|---|
1-2 | 9/15 AT-His-T7-RBS-His (1)-(2) |
3-7 | 9/16 AT-His-T7-RBS-His (1)-(5) |
8-9 | 9/16 dddD-T7-RBS (After mutation)(1)-(2) |
10-16 | 9/16 SAMmt-pSB1C3 (1)-(7) |
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|
9/17 REDOX-His (DNA purification product) | 5.5 | - | 1 | - | 1 | M | 3 | 3 | 16.5 | 30 |
9/17 REDOX (DNA purification product) | 4.6 | - | 1 | - | 1 | M | 3 | 3 | 17.4 | 30 |
Ligation
Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) | ||||
---|---|---|---|---|---|---|---|---|---|
9/16 T7-RBS(S,P) (DNA purification product) | 1.4 | 9/17 AT(X,P) (DNA purification product) | 2.3 | (x2)Buffer | 5 | T4 Ligase | 1 | 0.3 | 10 |
YCplac111(SmaI cut) | 1 | 9/17 REDOX-His (DNA purification product) | 0.6 | (x2)Buffer | 5 | T4 Ligase | 1 | 2.39 | 10 |
9/16 pSB1C3(E,P) (DNA purification product) | 0.8 | 9/17 SAMmt(E,P) (DNA purification product) | 1.9 | (x2)Buffer | 5 | T4 Ligase | 1 | 0.8 | 10 |
YCplac111(SmaI cut) | 1 | - | - | (x2)Buffer | 5 | T4 Ligase | 1 | 3 | 10 |
Transformation
Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium | ||
---|---|---|---|---|---|
9/17 AT-T7-RBS (Ligation product) | 2 | DH5α | 10 | 12 | SOC |
9/17 REDOX-His-YCplac111 (Ligation product) | 2 | DH5α | 10 | 12 | SOC |
9/17 SAMmt-pSB1C3 (Ligation product) | 2 | DH5α | 10 | 12 | SOC |
9/17 Control (SmaX3, Ligation product) | 2 | DH5α | 10 | 12 | SOC |
Make Control (for 9/18 UV-Spectrum)
Add MTOB (1.6mg) to ethanenitrile (313.4µL)
Electrophoresis
(1)
Lane | Sample | ||
---|---|---|---|
1-2 | 9/15 AT-His-T7-RBS His (1)(2) | ||
3-7 | 9/15 AT-His-T7-RBS His (1)-(5) | ||
8 | 9/16 dddD-T7-RBS(After mutation)(1) | ||
9 | 1kb ladder |
(2)
Lane | Sample | ||
---|---|---|---|
1 | 1kb ladder | ||
2 | 9/16 dddD-T7-RBS(After mutation)(2) | ||
3-8 | 9/16 SAMmt-pSB1C3 (1)-(6) |
(3)
Lane | Sample | ||
---|---|---|---|
1 | 1kb ladder | ||
2 | 9/16 SAMmt-pSB1C3 (7) |
Liquid Culture
Sample |
---|
9/16 AT His-T7-RBS His (2)-(4) |
9/16 SAMmt-pSB1C3 (2)(4) |
DNA Purification
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
9/17 REDOX His (X,P) | 1.6 | 1.74 | - |
9/17 REDOX (X,P)(PCR product) | 1.3 | 2.43 | - |
9/18
Miniprep
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
9/15 T7-AT-His (1) | 200 | 1.54 | 1.60 |
9/15 T7-AT-His (2) | 180 | 1.64 | 2.02 |
9/16 T7-AT-His (2) | 140 | 1.84 | 2.09 |
9/16 T7-AT-His (3) | 140 | 1.71 | 1.82 |
9/16 T7-AT-His (4) | 145 | 1.83 | 2.16 |
9/16 SAMmt-pSB1C3 (2) | 225 | 1.64 | 1.71 |
9/16 SAMmt-pSB1C3 (4) | 190 | 1.75 | 2.12 |
Colony PCR
Master Mix
Quick Taq/(µL) | Fw M13/(µL) | Rv M13/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
45 | 1.8 | 1.8 | 41.4 | 90 |
Predenature | Denature | Annealing | Extension | Cooling |
---|---|---|---|---|
94°C | 94°C | 55°C | 68°C | 12°C |
2min | 30sec | 30sec | 90sec | Infinity |
Denature-Annealing-Extension 30cycle
Number | Sample |
---|---|
1 | 9/16 REDOX-(1)-1 |
2 | 9/16 DiDECARBO-(1)-1 |
3 | 9/16 DiDECARBO-(1)-2 |
4 | 9/16 DiDECARBO-(1)-3 |
5 | 9/16 DiDECARBO-(1)-4 |
6 | 9/17 AT-T7-RBS-1 |
7 | 9/17 REDOX-His-YCplac111-1 |
8 | 9/17 REDOX-His-YCplac111-2 |
9 | 9/17 REDOX-His-YCplac111-3 |
Lane | Sample |
---|---|
1 | 1kb ladder |
2 | 9/16 REDOX-YCplac111 (1) |
3-6 | 9/16 DiDECARB-YCplac111 (1)-(4) |
Lane | Sample |
---|---|
1 | - |
2 | 1kb ladder |
3 | 9/17 AT-T7-RBS (1) |
4-6 | REDOX-His=YCplac111 (1)-(3) |
Liquid Culture
Sample | Medium |
---|---|
9/17 REDOX-His-YCplac111-(1) | CP |
9/17 REDOX-His-YCplac111-(2) | CP |
9/14 AT-pSB1C3-(1) | CP |
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|
9/15 AT-pSB1C3-(3) | 15 | 2 | - | - | 2 | H | 6 | 6 | 51.5 | 82.5 |
9/15 AT-pSB1C3-(2) | 19 | - | 2 | 2 | - | MC | 6 | 6 | 45 | 80 |
9/9 T7-RBS-pSB1C3 | 14 | - | - | 2 | 2 | B | 6 | 6 | 50 | 80 |
9/18 T7-RBS-AT-His-pSB1C3 | 33 | - | - | 3 | 3 | B | 9 | 9 | 63 | 120 |
UV-spequtrum measurement
Ethanenitrile solution of saturated MTOB (x10)Mutation PCR
SAMmt (SpeI)
Prime STAR MAX Premix(2x) | Fw SAMmt SpeI mutation primer | Rv SAMmt SpeI mutation primer | 9/18 SAMmt-pSB1C3-4 | MilliQ | Total |
---|---|---|---|---|---|
25µL | 1µL | 1µL | pick upµL | 23µL | 50µL |
Predenature | Denature | Annealing | Extension | Cooling |
---|---|---|---|---|
98°C | 98°C | 55°C | 72°C | 12°C |
5min | 10sec | 15sec | 30sec | Infinity |
Denature-Annealing-Extension 30cycle
Quality check of preligation
Sample | Concentration(ng/µL) | A260 | A280 | 260/280 | 260/230 |
---|---|---|---|---|---|
DiDECARB | 538.36 | 10.767 | 5.734 | 1.88 | 2.07 |
DECARB | 223.37 | 4.467 | 2.361 | 1.89 | 1.99 |
SAMmt-pSB1C3 | 185.24 | 3.705 | 1.896 | 1.95 | 1.87 |
REDOX | 454.07 | 9.081 | 4.800 | 1.89 | 2.17 |
AT-pSB1C3 | 299.73 | 5.995 | 2.993 | 2.00 | 1.99 |
T7-RBS | 306.31 | 6.126 | 2.901 | 2.11 | 1.97 |
T7-RBS-His | 307.59 | 6.152 | 3.129 | 1.97 | 1.50 |
Pcon-pSB1C3 | 245.88 | 4.918 | 2.423 | 2.03 | 1.65 |
PCR
DiDECARB | 10x Buffer | 2mM dNTPs | 25mM MgSO4 | #1 | #2 | KOD(1U/µL) | MilliQ | Total |
---|---|---|---|---|---|---|---|---|
1µL | 2.5µL | 2.5µL | 1.5µL | 1.25µL | 1.25µL | 0.5µL | 14.5µL | 25 |
25µL x4 (100µL)
(1)
#1: DiDECARB OE-A (10 µM)
#2: DiDECARB Mutation Rv (10 µM)
Predenature | Denature | Annealing | Extension | Cooling |
---|---|---|---|---|
94°C | 98°C | 50-65°C | 68°C | 15°C |
2min | 10sec | 30sec | 48sec | Infinity |
Denature-Annealing-Extension 30cycle
(2)
#1: DiDECARB OE-D (10 µM)
#2: DiDECARB Mutation Fw (10 µM)
(3)
#1: RV-DiDECARB His (10 µM)
#2: DiDECARB Mutation Fw (10 µM)
Predenature | Denature | Annealing | Extension | Cooling |
---|---|---|---|---|
94°C | 98°C | 50-65°C | 68°C | 15°C |
2min | 10sec | 30sec | 78sec | Infinity |
Denature-Annealing-Extension 30cycle
(4)
#1: DiDECARB OE-A (10 µM)
#2: DiDECARB MutationP Rv (10 µM)
(5)
#1: DiDECARB MutationP-Fw (10 µM)
#2: DiDECARB MutationE-Rv (10 µM)
Predenature | Denature | Annealing | Extension | Cooling |
---|---|---|---|---|
94°C | 98°C | 50-65°C | 68°C | 15°C |
2min | 10sec | 30sec | 48sec | Infinity |
Denature-Annealing-Extension 30cycle
(6)
#1: DiDECARB OE-D (10 µM)
#2: DiDECARB Mutation-E-Fw (10 µM)
(7)
#1: RV-DiDECARB His (10 µM)
#2: DiDECARB Mutation E-Fw (10 µM)
Predenature | Denature | Annealing | Extension | Cooling |
---|---|---|---|---|
94°C | 98°C | 50-65°C | 68°C | 15°C |
2min | 10sec | 30sec | 78sec | Infinity |
Denature-Annealing-Extension 30cycle
(8)
#1: SAMmt OE-A (10 µM)
#2: SAMmt Mutation-Rv (10 µM)
Predenature | Denature | Annealing | Extension | Cooling |
---|---|---|---|---|
94°C | 98°C | 50-65°C | 68°C | 15°C |
2min | 10sec | 30sec | 48sec | Infinity |
Denature-Annealing-Extension 30cycle
(9)
#1: SAMmt OE-D (10 µM)
#2: SAMmt Mutation-Fw (10 µM)
(10)
#1: RV-SAMmt His (10 µM)
#2: SAMmt Mutation-Fw (10 µM)
Predenature | Denature | Annealing | Extension | Cooling |
---|---|---|---|---|
94°C | 98°C | 50-65°C | 68°C | 15°C |
2min | 10sec | 30sec | 78sec | Infinity |
Denature-Annealing-Extension 30cycle
(11)
#1: Fw-Pre-REDPX (10 µM)
#2: Suf-REDOX-RV (10 µM)
(12)
#1: Fw-Pre-REDPX (10 µM)
#2: RV-REDOX-His (10 µM)
Predenature | Denature | Annealing | Extension | Cooling |
---|---|---|---|---|
94°C | 98°C | 50-65°C | 68°C | 15°C |
2min | 10sec | 30sec | 48sec | Infinity |
Denature-Annealing-Extension 30cycle
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | XhoI | DraI | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|---|---|
AT-pSB1C3 (299ng/µL) | 4 | - | 1 | - | 1 | 1 | 1 | M | 5 | 5 | 32 | 50 |
AT-pSB1C3 (299ng/µL) | 6.8 | - | 1 | - | 1 | - | - | M | 3 | 3 | 15.20 | 30 |
pSB1C3-T7-RBS (310ng/µL) | 7 | - | - | 1 | 1 | - | - | H | 5 | 1* | 35 | 50 |
Pcon-pSB1C3 (245ng/µL) | 4 | 1 | - | - | 1 | - | - | H | 5 | 1* | 38 | 50 |
The cell marked * is not BSA but BAP.
9/19
Sequence PCR
Number/(µL) | Sample | Template/(µL) | BigDye/(µL) | 5x Buffer/(µL) | Primer/(µL) | MilliQ/(µL) | Conc/(µL) | ||||
---|---|---|---|---|---|---|---|---|---|---|---|
1 | T7-RBS-AT-His(9/16-(2)) | 2.8 | 0.5 | 1.75 | VF | 0.5 | 4.95 | 10.5 | |||
2 | T7-RBS-AT-His(9/16-(3)) | 2.8 | 0.5 | 1.75 | VF | 0.5 | 4.95 | 10.5 | |||
3 | SAM-pSB1C3(4) | 2.8 | 0.5 | 1.75 | VF | 0.5 | 4.95 | 10.5 | |||
4 | T7-RBS-AT-His(9/16-(2)) | 2.8 | 0.5 | 1.75 | VR | 0.5 | 4.95 | 10.5 | |||
5 | T7-RBS-AT-His(9/16-(3)) | 2.8 | 0.5 | 1.75 | VR | 0.5 | 4.95 | 10.5 | |||
6 | SAM-pSB1C3(4) | 2.8 | 0.5 | 1.75 | VR | 0.5 | 4.95 | 10.5 | |||
7 | T7-RBS-AT-His(9/16-(2)) | 2.8 | 0.5 | 1.75 | seg-1 | 0.5 | 4.95 | 10.5 | |||
8 | T7-RBS-AT-His(9/16-(3)) | 2.8 | 0.5 | 1.75 | seg-1 | 0.5 | 4.95 | 10.5 | |||
9 | SAM-pSB1C3(4) | 2.8 | 0.5 | 1.75 | OE-B | 0.5 | 4.95 | 10.5 | |||
10 | T7-RBS-AT-His(9/16-(2)) | 2.8 | 0.5 | 1.75 | seg-2 | 0.5 | 4.95 | 10.5 | |||
11 | 9/23M PQLB | 2.8 | 0.5 | 1.75 | seg-2 | 0.5 | 4.95 | 10.5 |
Predenature | Denature | Annealing | Extension | Cooling | Cooling |
---|---|---|---|---|---|
96°C | 96°C | 50°C | 60°C | 4°C | 4°C |
2min | 10sec | 5sec | 4min | 1min | infinity |
Denature-Annealing-Extension 30cycle
Gel Extraction
Lane | Sample |
---|---|
1,2,4,5,8 | 1kbp ladder |
3 | T7-RBS-AT-His-pSB1C3 S,P |
6 | T7-RBS-pSB1C3 S,P |
Miniprep
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
9/18 T7-RBS-AT-His(2) | 305 | 1.66 | 1.49 |
9/18 T7-RBS-His-pSB1C3(1) | 280 | 1.68 | 1.67 |
9/18 REDOX(His)-YC Plac3(1) | 985 | 1.74 | 2.39 |
9/18 REDOX(His)-YC Plac3(2) | 1025 | 1.72 | 2.28 |
Gel Extraction
Lane | Sample |
---|---|
1,2 | 9/18 AT-pSB1C3 X,S |
3 | 100bp ladder |
5,6 | 9/18 AT-pSB1C3 E,P |
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|
9/19 T7-RBS-His Miniprep product | 20 | - | - | 3 | 3 | B | 12 | 0 | 82 | 120 |
9/19 REDOX-3 M(1) | 10.1 | 5 | - | - | 5 | H | 20 | 0 | 159.9 | 200 |
9/19 REDOX-3 M(1) control | 0.1 | - | - | - | - | H | 1 | - | 8.9 | 10 |
Electrophoresis
Lane | Sample |
---|---|
1 | DiDECARB-OE-A 52.1°C |
2 | DiDECARB-OE-A 55.4°C |
3 | DiDECARB-OE-A 59.4°C |
4 | DiDECARB-OE-A 64.9°C |
5 | DiDECARB-OE-A 52.1°C |
6 | DiDECARB-OE-A 55.4°C |
7 | DiDECARB-OE-A 59.4°C |
8 | DiDECARB-OE-A 64.9°C |
9 | DiDECARB-Mutation PCR product Pst1 Fwd 52.1°C |
10 | DiDECARB-Mutation PCR product Pst1 Fwd 55.4°C |
11 | DiDECARB-Mutation PCR product Pst1 Fwd 59.4°C |
12 | DiDECARB-Mutation PCR product Pst1 Fwd 64.9°C |
13 | REDOX-Fwd 52.1°C |
14 | REDOX-Fwd 55.4°C |
15 | REDOX-Fwd 59.4°C |
16 | REDOX-Fwd 64.9°C |
17 | REDOX-Fwd-His 52.1°C |
18 | REDOX-Fwd-His 55.4°C |
19 | REDOX-Fwd-His 59.4°C |
20 | REDOX-Fwd-His 64.9°C |
21 | DiDECARB-Mutation PCR product Fwd-His 52.1°C |
22 | DiDECARB-Mutation PCR product Fwd-His 55.4°C |
23 | DiDECARB-Mutation PCR product Fwd-His 59.4°C |
24 | DiDECARB-Mutation PCR product Fwd-His 64.9°C |
25 | DiDECARB-Mutation PCR product Fwd 52.1°C |
26 | DiDECARB-Mutation PCR product Fwd 55.4°C |
27 | DiDECARB-Mutation PCR product Fwd 59.4°C |
28 | DiDECARB-Mutation PCR product Fwd 64.9°C |
29 | SAM-Mutation PCR product Fwd 52.1°C |
30 | SAM-Mutation PCR product Fwd 55.4°C |
31 | SAM-Mutation PCR product Fwd 59.4°C |
32 | SAM-Mutation PCR product Fwd 64.9°C |
33 | 1kbp ladder |
34 | SAM-Mutation PCR product Fwd 52.1°C |
35 | SAM-Mutation PCR product Fwd 55.4°C |
36 | SAM-Mutation PCR product Fwd 59.4°C |
37 | SAM-Mutation PCR product Fwd 64.9°C |
38 | SAM-Mutation PCR product Fwd-His 52.1°C |
39 | SAM-Mutation PCR product Fwd-His 55.4°C |
40 | SAM-Mutation PCR product Fwd-His 59.4°C |
41 | SAM-Mutation PCR product Fwd-His 64.9°C |
42 | 1kbp ladder |
43 | DiDECARB-Mutation PCR product E-His 52.1°C |
44 | DiDECARB-Mutation PCR product E-His 55.4°C |
45 | DiDECARB-Mutation PCR product E-His 59.4°C |
46 | DiDECARB-Mutation PCR product E-His 64.9°C |
47 | DiDECARB-Mutation PCR product E-Fwd 52.1°C |
48 | DiDECARB-Mutation PCR product E-Fwd 55.4°C |
49 | DiDECARB-Mutation PCR product E-Fwd 59.4°C |
50 | DiDECARB-Mutation PCR product E-Fwd 64.9°C |
PCR
Template;DiDECARB-OE-A,DiDECARB Mutation product Fw
10x Buffer/(µL) | 2.0mM dNTP/(µL) | 25.mM MgSO4/(µL) | 10.0µM DiDECARB-OE-A/(µL) | 10.0µM DiDECARB-OE-D/(µL) | 1.0U/µL KOD/(µL) | H2O | Total/(µL) |
---|---|---|---|---|---|---|---|
10 | 10 | 6 | 5 | 5 | 2 | up tp 100 | 100 |
Template;DiDECARB-OE-A,DiDECARB Mutation product Fw-His
10x Buffer/(µL) | 2.0mM dNTP/(µL) | 25.mM MgSO4/(µL) | 10.0µM DiDECARB-OE-A/(µL) | 10.0µM Rv DiDECARB-His/(µL) | 1.0U/µL KOD/(µL) | H2O | Total/(µL) |
---|---|---|---|---|---|---|---|
10 | 10 | 6 | 5 | 5 | 2 | up tp 100 | 100 |
Template;DiDECARB-OE-A,DiDECARB Mutation produc Pst1t Fw,DiDECARB Mutation produc Eco1
10x Buffer/(µL) | 2.0mM dNTP/(µL) | 25.mM MgSO4/(µL) | 10.0µM DiDECARB-OE-A/(µL) | 10.0µM DiDECARB-OE-D/(µL) | 1.0U/µL KOD/(µL) | H2O | Total/(µL) |
---|---|---|---|---|---|---|---|
10 | 10 | 6 | 5 | 5 | 2 | up tp 100 | 100 |
Template;DiDECARB-OE-A,DiDECARB Mutation product Pst1 Fw,DiDECARB Mutation product Eco1-His
10x Buffer/(µL) | 2.0mM dNTP/(µL) | 25.mM MgSO4/(µL) | 10.0µM DiDECARB-OE-A/(µL) | 10.0µM Rv DiDECARB-His/(µL) | 1.0U/µL KOD/(µL) | H2O | Total/(µL) |
---|---|---|---|---|---|---|---|
10 | 10 | 6 | 5 | 5 | 2 | up tp 100 | 100 |
Template;SAM-OE-A,SAM Mutation product Fw
10x Buffer/(µL) | 2.0mM dNTP/(µL) | 25.mM MgSO4/(µL) | 10.0µM SAM-OE-A/(µL) | 10.0µM SAM-OE-D/(µL) | 1.0U/µL KOD/(µL) | H2O | Total/(µL) |
---|---|---|---|---|---|---|---|
10 | 10 | 6 | 5 | 5 | 2 | up tp 100 | 100 |
Template;SAM-OE-A,SAM Mutation product Fw-His
10x Buffer/(µL) | 2.0mM dNTP/(µL) | 25.mM MgSO4/(µL) | 10.0µM SAM-OE-A/(µL) | 10.0µM SAM His/(µL) | 1.0U/µL KOD/(µL) | H2O | Total/(µL) |
---|---|---|---|---|---|---|---|
10 | 10 | 6 | 5 | 5 | 2 | up tp 100 | 100 |
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
pSB1C3-AT/Pst1,Xba1,Xhol1,Dra1 | 24.75 | 1.90 | 1.92 |
T7RBS/Spe1,Pst1 | 8.48 | 1.91 | 1.01 |
T7RBS-His/Spe1,Pst1 | 35.98 | 1.88 | 1.78 |
Gel Extraction/Electrophoresis
Lane | Sample |
---|---|
2~5 | DiDECARB His |
6~8,10 | DiDECARB |
11~14 | SAM |
16~20 | SAM His |
21~23,25 | DE His |
26~30 | DE |
1,9,15,24 | Blank |
DNA Purification
DiDECARB His
DiDECARB
SAM
SAM His
Restriction Enzyme Digestion
Sample/(ng) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|
DiDECARB His X,P | 2000 | - | 1 | - | 1 | M | 3 | 3 | up tp 30 | 30 |
DiDECARB X,P | 2000 | - | 1 | - | 1 | M | 3 | 3 | up to 30 | 30 |
DiDECARB E,P | 2000 | 1 | - | - | 1 | H | 3 | - | up to 30 | 30 |
SAM X,P | 2000 | - | 1 | - | 1 | M | 3 | 3 | - | 30 |
SAM E,P | 2000 | 1 | - | - | 1 | H | 3 | - | up to 30 | 30 |
SAM His X,P | 2000 | - | 1 | - | 1 | M | 3 | 3 | up to 30 | 30 |
9/20
Electrophoresis
Lane | Sample |
---|---|
1 | SAM His X,P |
2 | Di X,P |
3 | Di His X,P |
4 | SAM E,P |
5 | SAM X,P |
6 | Di E,P |
7 | AT X,P 2 |
Lane | Sample |
---|---|
1 | AT X,P 3 |
2 | T7-RBS S,P |
3 | dddD Mutation PCR product (2) |
4 | dddD Mutation PCR product (1) |
5 | T7-RBS pSB1C3 |
6 | REDOX PCR product |
7 | REDOX His PCR product |
Transformation
Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium | ||
---|---|---|---|---|---|
7-2 DiDECARB T7-RBS | 2 | DH5α | 10 | 12 | SOC |
7-5 SAMmt T7-RBS | 2 | DH5α | 10 | 12 | SOC |
7-11 T7-RBS dddD(1) Mutation PCR product | 2 | DH5α | 10 | 12 | SOC |
7-12 T7-RBS dddD(3) Mutation PCR product | 2 | DH5α | 10 | 12 | SOC |
7-13 T7-RBS AT | 2 | DH5α | 10 | 12 | SOC |
T7-RBS control | 2 | DH5α | 10 | 12 | SOC |
10-1 DiDECARB pSB1C3 | 2 | DH5α | 10 | 12 | SOC |
10-4 SAMmt pSB1C3 | 2 | DH5α | 10 | 12 | SOC |
10-8 REDOX pSB1C3 | 2 | DH5α | 10 | 12 | SOC |
pSB1C3 control | 2 | DH5α | 10 | 12 | SOC |
14-3 T7-RBS SAM-His | 2 | DH5α | 10 | 12 | SOC |
T7-RBS DiDECARB His | 2 | DH5α | 10 | 12 | SOC |
REDOX T7 RBS His(14)-9 | 2 | DH5α | 10 | 12 | SOC |
T7-RBS-His control | 2 | DH5α | 10 | 12 | SOC |
9/22
Colony PCR
Quick Tag/(µL) | VF2/(µL) | VR/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
5 | 0.2 | 0.2 | 4.6 | 10 |
Predenature | Denature | Annealing | Extension | Cooling |
---|---|---|---|---|
94°C | 94°C | 55°C | 68°C | 12°C |
2min | 30sec | 30sec | 150sec | infinity |
Denature-Annealing-Extension 30cycle
Number | Sample |
1~8 | REDOX-T7-RBS-His |
---|---|
9~16 | T7-RBS-SAM-His |
17~24 | T7-RBS control |
25~32 | T7-RBS-dddD |
33~40 | DiDECARB-pSB1C3 |
41~48 | pSB1C3 control |
49~56 | T7-RBS-DiDECARB |
57~64 | T7-RBS-His |
65~72 | REDOX-pSB1C3 |
73~80 | DiDECARB-T7-RBS |
81~88 | SAM-pSB1C3 |
89~96 | T7-RBS-sssS |
97~104 | T7-RBS-SAM |
105 | T7-RBS-AT |
106~123 | pBC345-AT |
Colony PCR
Master Mix
Quick Tag/(µL) | VF2/(µL) | VR/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
225 | 9 | 9 | 217 | 450 |
Predenature | Denature | Annealing | Extension |
---|---|---|---|
94°C | 94°C | 55°C | 68°C |
2min | 30sec | 30sec | 150sec |
Denature-Annealing-Extension 30cycle
Number | Sample |
---|---|
1~7 | 9/20 T7-RBS-REDOX-His |
8~14 | 9/20 SAMmt-pSB1C3 |
15~21 | 9/20 T7-RBS-SAMmt-His |
22~28 | 9/20 DiDECARB-pSB1C3 |
29~35 | 9/20 T7-RBS-DiDECARB |
36~42 | 9/20 T7-RBS-DiDECARB-His |
43 | 9/20 T7-RBS-dddD |
44 | 9/20 DiDECARB-pSB1C3 |
Miniprep
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
T7-RBS-His | 55 | 1.58 | 0.80 |
Concentration Measurement
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
T7-RBS-His S,P | 1.0 | 1.54 | 1.94 |
T7-RBS-His S,P | 1.3 | 1.59 | 2.01 |
T7-RBS E,S | 1.1 | 1.40 | 2.01 |
HT-pSB1C3 X,P | 4.4 | 1.52 | 1.08 |
AT-pSB1C3 X,P | 4.0 | 1.65 | 1.06 |
Cut Check
Lane | Sample |
---|---|
1 | Blank |
2 | 100bp GeneRuler |
3 | T7 RBS-His S,P |
4 | T7-RBS S,P |
5 | T7-RBS E,P |
6 | AT-pSB1C3 X,P |
7 | AT-pSB1C3 X,P |
Electrophoresis
(1)
Lane | Sample |
---|---|
1 | 100b ladder |
2~8 | T7-RBS-REDOX-His |
(2)
Lane | Sample |
---|---|
1 | 100b ladder |
2~8 | SAMmt-pSB1C3 |
(3)
Lane | Sample |
---|---|
1 | 100b ladder |
2~8 | T7-RBS-SAMmt-His |
(4)
Lane | Sample |
---|---|
1 | 100b ladder |
2~8 | DiDECARB-pSB1C3 |
(5)
Lane | Sample |
---|---|
1 | 100b ladder |
2~8 | T7-RBS-DiDECARB |
(6)
Lane | Sample |
---|---|
1 | 100b ladder |
2~8 | T7-RBS-DiDECARB-His |
(7)
Lane | Sample |
---|---|
1 | Blank |
2 | Blank |
3 | 100b ladder |
4 | T7-RBS-dddD-1 |
5 | DiDECARB-pSB1C3-1 |
Liquid Culture
Sample | Medium |
---|---|
T7-RBS-dddD | CP |
Sample | Medium |
---|---|
REDOX-pSB1C3 | CP |
Sample | Medium |
---|---|
T7-RBS-SAMmt | CP |
Colony PCR
Master Mix
Quick Taq/(µL) | VF2/(µL) | VR/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
240 | 9.6 | 9.6 | 220.8 | 480 |
Predenature | Denature | Annealing | Extension |
---|---|---|---|
94°C | 94°C | 55°C | 68°C |
2min | 30sec | 30sec | 90sec |
Denature-Annealing-Extension 30cycle
Number | Sample |
---|---|
1~16 | T7-RBS-DiDECARB-His |
19~32 | T7-RBS-SMAmt-His |
33~48 | T7-RBS-REDOX-His |
9/23
Miniprep
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
pB-22 | 250 | 1.61 | 1.69 |
pB-23 | 125 | 2.07 | 2.90 |
pQ-3 | 185 | 1.68 | 1.87 |
pQ-5 | 210 | 1.72 | 1.86 |
pLQB | 90 | 2.40 | 3.09 |
Electrophoresis
1
Number | Sample |
---|---|
1 | 100bp ladder |
2~8 | T7-RBS-REDOX-His 2~7 |
2
Number | Sample |
---|---|
1 | 100bp ladder |
2~8 | T7-RBS-REDOX-His 8~14 |
3
Number | Sample |
---|---|
1 | 100bp ladder |
2,3 | T7-RBS-REDOX-His 15,16 |
4~8 | T7-RBS-SAMmt-His 1~5 |
4
Number | Sample |
---|---|
1 | 100bp ladder |
2~8 | T7-RBS-SAMmt-His 6~12 |
5
Number | Sample |
---|---|
1 | 100bp ladder |
2~5 | T7-RBS-SAMmt-His 13~16 |
6~8 | T7-RBS-DiDECARB-His 1~3 |
6
Number | Sample |
---|---|
1 | 100bp ladder |
2~8 | T7-RBS-DiDECARB-His 4~10 |
7
Number | Sample |
---|---|
1 | 100bp ladder |
2~7 | T7-RBS-DiDECARB-His 11~16 |
9/24
Dephosphorylation
T7-RBS-His S,P
Miniprep
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
T7-RBS-SAM 6 | 10.3 | 1.67 | 1.15 |
9/22 DiDECARB pSB1C3-4 | 11.3 | 1.54 | 1.11 |
9/22 DiDECARB pSB1C3-2 | 10.5 | 1.67 | 0.83 |
9/20 T7-RBS-dddD Mutation PCR product(3)-(4) | 14.2 | 1.71 | 1.38 |
REDOX-pSB1C3(2) | 9.8 | 1.71 | 0.83 |
9/20 T7-RBS-dddD Mutation PCR product(3)-(5) | 12.7 | 1.66 | 1.42 |
9/22 DiDECARB pSB1C3-5 | 10.5 | 1.69 | 1.15 |
Cycle PCR
Master Mix
Number/(µL) | Sample | Template/(µL) | BigDye/(µL) | 5x Buffer/(µL) | Primer/(µL) | MilliQ/(µL) | Conc/(µL) | |
---|---|---|---|---|---|---|---|---|
1 | 9/23M PQLB | 4.4 | 0.5 | 1.75 | VF2 | 0.5 | 2.8 | 90 |
2 | 9/23M PQLB | 4.4 | 0.5 | 1.75 | pQLB seq-1 | 0.5 | 2.8 | 90 |
3 | 9/23M PQLB | 4.4 | 0.5 | 1.75 | pQLB seq-2 | 0.5 | 2.8 | 90 |
4 | 9/23M PQLB | 4.4 | 0.5 | 1.75 | pQLB seq-3 | 0.5 | 2.8 | 90 |
5 | 9/23M PQLB | 4.4 | 0.5 | pQLB seq-4 | VF2 | 0.5 | 2.8 | 90 |
6 | 9/23M PQLB | 4.4 | 0.5 | 1.75 | VR | 0.5 | 2.8 | 90 |
7 | 9/23M PQ-3 | 2.2 | 0.5 | 1.75 | VF2 | 0.5 | 5.0 | 185 |
8 | 9/23M PQ-3 | 2.2 | 0.5 | 1.75 | VR | 0.5 | 5.0 | 185 |
9 | 9/23M PQ-5 | 1.9 | 0.5 | 1.75 | VF2 | 0.5 | 5.3 | 210 |
10 | 9/23M PQ-5 | 1.9 | 0.5 | 1.75 | VR | 0.5 | 5.3 | 210 |
11 | 9/22M pB-23 | 3.2 | 0.5 | 1.75 | VF2 | 0.5 | 4.0 | 125 |
12 | 9/22M pB-23 | 3.2 | 0.5 | 1.75 | VR | 0.5 | 4.0 | 125 |
13 | 9/23M pB-22 | 1.6 | 0.5 | 1.75 | VF2 | 0.5 | 5.7 | 250 |
14 | 9/23M pB-22 | 1.6 | 0.5 | 1.75 | VR | 0.5 | 5.7 | 250 |
15 | 9/15 MP AT-pSB1C3 1 | 1.3 | 0.5 | 1.75 | VF2 | 0.5 | 6.0 | 299 |
16 | 9/15 MP AT-pSB1C3 1 | 1.3 | 0.5 | 1.75 | AT Seq1 | 0.5 | 6.0 | 299 |
17 | 9/15 MP AT-pSB1C3 1 | 1.3 | 0.5 | 1.75 | AT Seq2 | 0.5 | 6.0 | 299 |
18 | 9/15 MP AT-pSB1C3 1 | 1.3 | 0.5 | 1.75 | VR | 0.5 | 6.0 | 299 |
19 | 9/22 M REDOX pSB1C3 | 0.8 | 0.5 | 1.75 | VF2 | 0.5 | 6.4 | 490 |
20 | 9/22 M REDOX pSB1C3 | 0.8 | 0.5 | 1.75 | REDOX Seq1 | 0.5 | 6.4 | 490 |
21 | 9/22 M REDOX pSB1C3 | 0.8 | 0.5 | 1.75 | VR | 0.5 | 6.4 | 490 |
22 | 9/18 MP SAM-pSB1C3 | 2.1 | 0.5 | 1.75 | VF2 | 0.5 | 5.0 | 185 |
23 | 9/18 MP SAM-pSB1C3 | 2.1 | 0.5 | 1.75 | SAM Seq1 | 0.5 | 5.0 | 185 |
24 | 9/18 MP SAM-pSB1C3 | 2.1 | 0.5 | 1.75 | VR | 0.5 | 5.0 | 185 |
25 | 9/22 DiDECARB pSB1C3-4 | 0.7 | 0.5 | 1.75 | VF | 0.5 | 6.5 | 565 |
26 | 9/22 DiDECARB pSB1C3-4 | 0.7 | 0.5 | 1.75 | DiDECARB Seq1 | 0.5 | 6.5 | 565 |
27 | 9/22 DiDECARB pSB1C3-4 | 0.7 | 0.5 | 1.75 | DiDECARB Seq2 | 0.5 | 6.5 | 565 |
28 | 9/22 DiDECARB pSB1C3-4 | 0.7 | 0.5 | 1.75 | VR | 0.5 | 6.5 | 565 |
DNA Purification
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
9/24 PCR product AT | 265 | 1.67 | 2.10 |
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|
9/24 REDOX(His) column | 7.5 | - | 1 | - | 1 | M | 3 | 3 | 14.5 | 30 |
9/25
Colony PCR
Quick Taq/(µL) | VF2/(µL) | VR/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
5 | 0.2 | 0.2 | 4.6 | 10 |
Predenature | Denature | Annealing | Extension | Cooling |
---|---|---|---|---|
94°C | 94°C | 55°C | 68°C | 12°C |
2min | 30sec | 30sec | 1min | infinity |
Denature-Annealing-Extension 30cycle
Number | Sample |
---|---|
1 | T7-RBS-REDOX-His(1) |
2 | T7-RBS-REDOX-His(2) |
3 | T7-RBS-REDOX-His(3) |
Electrophoresis
Lane | Sample |
---|---|
1,2 | Blank |
3 | 100b ladder |
4~6 | T7-RBS-REDOX-His(1)~(3) |
Colony PCR
Master Mix
Quick Taq/(µL) | VF2/(µL) | VR/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
130 | 5.2 | 5.2 | 119.6 | 260.0 |
Predenature | Denature | Annealing | Extension |
---|---|---|---|
94°C | 94°C | 55°C | 68°C |
2min | 30sec | 30sec | 90sec |
Denature-Annealing-Extension 30cycle
Number | Sample |
---|---|
1~8 | T7-RBS-REDOX-His (1)~(8) |
9~16 | T7-RBS-SAMmt-His (1)~(8) |
17~24 | T7-RBS-DiDECARB-His (1)~(8) |
Electrophoresis
1
Lane | Sample |
---|---|
1 | 100bp GeneRuler |
2~8 | T7-RBS-REDOX-His(1)~(7) |
2
Lane | Sample |
---|---|
1 | 100bp GeneRuler |
2 | T7-RBS-REDOX-His(8) |
3~7 | T7-RBS-SAMmt-His(1)~(5) |
8 | Blank |
3
Lane | Sample |
---|---|
1 | 100bp GeneRuler |
2~4 | T7-RBS-SAMmt-His(6)~(8) |
5~8 | T7-RBS-DiDECARB-His(1)~(4) |
4
Lane | Sample |
---|---|
1 | 100bp GeneRuler |
2,3,5,6 | T7-RBS-DiDECARB-His(5)~(8) |
4 | Blank |
Transformation
Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium | ||
---|---|---|---|---|---|
T7-RBS-AT-His 9/19 M(3) | 1 | Zip BL21(DE3) | 10 | 11 | CP |
T7-RBS-dddD(n) 9/22 M (3)-5 | 1 | Zip BL21(DE3) | 10 | 11 | CP |
9/26
Colony PCR
Quick Taq/(µL) | VF2/(µL) | VR/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
5 | 0.2 | 0.2 | 4.6 | 10 |
Predenature | Denature | Annealing | Extension | Cooling |
---|---|---|---|---|
94°C | 94°C | 55°C | 68°C | 12°C |
5min | 30sec | 30sec | 2min | infinity |
Denature-Annealing-Extension 30cycle
Number | Sample |
---|---|
1~8 | T7-RBS-REDOX-His |
9~16 | T7-RBS-SAMmt-His |
17~24 | T7-RBS-DiDECARB-His |
Electrophoresis
Lane | Sample |
---|---|
1~8 | T7-RBS-REDOX-His(1)~(8) |
9~12,14~17 | T7-RBS-SAMmt-His(1)~(8) |
18~25 | T7-RBS-DiDECARB-His(1)~(8) |
13 | 100bp ladder |
9/27
Colony PCR
Quick Taq/(µL) | VF2/(µL) | VR/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
5 | 0.2 | 0.2 | 4.6 | 10 |
Predenature | Denature | Annealing | Extension |
---|---|---|---|
94°C | 94°C | 55°C | 68°C |
2min | 30sec | 30sec | 160sec |
Denature-Annealing-Extension 30cycle
Number | Sample |
---|---|
1~7 | T7-RBS-REDOX-His |
8~14 | T7-RBS-SAMmt-His |
15~20 | T7-RBS-DiDECARB-His |
21,22 | T7-RBS-AT-His 9/25 Transformation(3),(4) |
23,24 | T7-RBS-dddD 9/25 Transformation(3),(4) |
Cloning PCR
Master Mix
KOD Plus/(µL) | DNA CCAP 1023/11/(µL) | 2mMdNTPs/(µL) | 10xBuffer forKDDplusV2/(µL) | 25mM MgSO4/(µL) | Fw AT cloning primer 61/(µL) | Rv AT cloning primer 61/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|---|---|---|
1 | 2 | 5 | 5 | 3 | 1.5 | 1.5 | 31 | 50 |
Predenature | Denature | Annealing | Extension | Cooling |
---|---|---|---|---|
94°C | 98°C | 56°C | 68°C | 12°C |
2min | 10sec | 30sec | 90sec | infinity |
Denature-Annealing-Extension 30cycle
9/28
OE-PCR 1st
KOD Plus/(µL) | 9/27 AT PCR product/(µL) | 10xBuffer for KODPlus/(µL) | 2mMdNTPS/(µL) | 25mM MgSO4/(µL) | Primer #1/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|---|---|
1 | pick up | 5 | 5 | 3 | 1.5(each) | up to 50 | 50 |
1. #1 AT OE-A/AT OE-B
2. #1 AT OE-C/AT2 OE-B
3. #1 AT2 OE-C/AT OE-D
Predenature | Denature | Annealing | Extension | Final Extension | Cooling |
---|---|---|---|---|---|
98°C | 98°C | 55°C | 72°C | 72°C | 12°C |
2min | 10sec | 10sec | 1min | 5min | infinity |
Denature-Annealing-Extension 25cycle
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|
8/27 Miniprep product 1A2 | 7.6 | - | - | 3 | 3 | B | 12 | 1.2 | 93.2 | 120 |
control | 0.5 | - | - | - | - | B | 1 | 0.1 | 8.4 | 10 |
Gel Extraction
Lane | Sample |
---|---|
1 | 9/28 OE-(3) |
2 | 9/28 OE-(3) |
3 | Blank |
4 | 9/28 OE-(1) |
5 | 100bp ladder |
Lane | Sample |
---|---|
1 | 9/28 OE-(3) |
4 | 9/28 OE-(2) |
6 | 100bp ladder |
2,3,5 | Blank |
OE-PCR 2nd
Master Mix
KOD Plus/(µL) | 10xBuffer for KODPlus ver2/(µL) | 2mMdNTPS/(µL) | 25mM MgSO4/(µL) | Primer #1/(µL) | Template #2/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|---|---|
1 | 5 | 5 | 3 | 2(each) | 0.5(each) | up to 50 | 50 |
1. #1 AT OE-A/AT OE-D #2 9/28 OE-(1) gel extraction product/9/28 OE-(2) gel extraction product/9/28 OE-(3) gel extraction product
2. #1 AT OE-A/AT OE-D #2 9/27 AT PCR product 9/28 column
Predenature | Denature | Annealing | Extension | Final Extension | Cooling |
---|---|---|---|---|---|
98°C | 98°C | 55°C | 72°C | 72°C | 12°C |
2min | 10sec | 10sec | 1min | 5min | infinity |
Denature-Annealing-Extension 25cycle
9/29
Mutation PCR
PrimeStar Max Premix/(µL) | SAMmt Mutation Fw/(µL) | SAMmt Mutation Rv/(µL) | 9/18 MP SAM-pSB1C3/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
25 | 1 | 1 | 0.5 | 22.5 | 50 |
Denature | Annealing | Extension |
---|---|---|
98°C | 55°C | 72°C |
10sec | 15sec | 55sec |
Denature-Annealing-Extension 30cycle
Gel Extraction
Lane | Sample |
---|---|
5 | 100bp ladder |
3,7 | 1A2 9/28 S,P |
9 | 9/28 Digestion control |
1,2,4,5,6,8 | Blank |
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|
8/27 M 1A2 | 7.6 | - | 3 | - | 3 | D | 12 | 1.2 | 93.2 | 10 |
control | 0.5 | - | - | - | - | D | 1 | 0.1 | 8.4 | 10 |
Gel Extraction
Lane | Sample |
---|---|
1 | 100bp ladder |
4 | 1A2 9/29 X,P |
6 | 9/29 Digestion control |
2,3,5 | Blank |
Concentration Measurement
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
1. 9/28 OE(1) 9/29 gel extraction product | 17.9 | 1.92 | 0.94 |
2. 9/28 OE(2) 9/29 gel extraction product | 122.2 | 1.86 | 1.64 |
3. 9/28 OE(3) 9/29 gel extraction product | 78.8 | 1.89 | 1.67 |
1A2 9/29 X,P | 26.1 | 1.37 | 0.76 |
Transformation
Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium | ||
---|---|---|---|---|---|
9/29 Ligation DiDECARB(His)-1A2 | 1 | Zip BL21(DE3) | 10 | 11 | CP |
9/29 Ligation REDOX(His)-1A2 | 1 | Zip BL21(DE3) | 10 | 11 | CP |
9/29 SAMmt mutation | 1 | Zip BL21(DE3) | 10 | 11 | CP |
9/30
Mutation PCR
PrimeStar Max PreMix/(µL) | Template #1/(µL) | Primers #2/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
25 | 0.5 | 1(each) | up to 50 | 50 |
1. #1 9/15 Miniprep AT-pSB1C3(1) #2 Fw:AT Mutation Insert/Rv:AT Mutation Insert
2. #1 9/19 Miniprep T7-RBS-AT(His) #2 Fw Add His to T7RBS/Rv Add His toT7 RBS
3. #1 9/13 Miniprep T7-RBS-pSB1C3 #2 Fw Add His to T7RBS/Rv Add His to T7RBS
Denature | Annealing | Extension | Cooling |
---|---|---|---|
98°C | 55°C | 72°C | 12°C |
10sec | 15sec | 20sec | infinity |
Denature-Annealing-Extension 35cycle
DNA Purification
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
SAMmt-pSB1C3 9/30 column | 136.3 | 1.87 | 1.68 |
OE-PCR 2nd
Master Mix
KOD Plus/(µL) | 10xBuffer for KODPlus ver2/(µL) | 2mMdNTPS/(µL) | 25mM MgSO4/(µL) | Primer #1/(µL) | Template #2/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|---|---|
1 | 5 | 5 | 3 | 2(each) | 0.5(each) | up to 50 | 50 |
1. #1 AT OE-A/AT OE-D #2 9/28 OE-(1) gel extraction product/9/28 OE-(2) gel extraction product/9/28 OE-(3) gel extraction product
2. #1 AT OE-A/AT OE-D #2 9/27 AT PCR product 9/28 column
Predenature | Denature | Annealing | Extension | Final Extension | Cooling |
---|---|---|---|---|---|
98°C | 98°C | 55°C | 72°C | 72°C | 12°C |
2min | 10sec | 10sec | 1min | 5min | infinity |
Denature-Annealing-Extension 30cycle
Transformation
Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium | ||
---|---|---|---|---|---|
T7-RBS-AT-His | 1 | self-making DH5α | 10 | 11 | CP |
SAMmt-pSB1C3 9/29 Mutation PCR product | 1 | self-making DH5α | 10 | 11 | CP |
AT-pSB1C3 9/30 insertion Mutation PCR product | 1 | self-making DH5α | 10 | 11 | CP |
T7-RBS-His 9/30 Mutation PCR product | 1 | self-making DH5α | 10 | 11 | CP |
DNA Purification
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
9/30 OE-PCR 2nd product(Pre-AT-Suf) | 217 | 1.85 | 1.22 |
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|
Pre-At-Suf 9/30 Column | 9.2 | 1 | - | - | 1 | H | 4 | 0.4 | 14.4 | 40 |
10/1
DNA Purification
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
AT 9/30 E,P | 38 | 1.84 | 0.46 |
pSB1C3 E,P | 5 | 1.78 | 0.27 |
Transformation
Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium | ||
---|---|---|---|---|---|
9/30 AT-pSB1C3 | 1 | BL21(DE3) | 10 | 11 | CP |
9/30 T7-RBS-His | 1 | self-making DH5α 2/23 | 10 | 11 | CP |
9/30 T7-RBS-AT-His | 1 | self-making DH5α 2/23 | 10 | 11 | CP |
9/29 SAMmt-pSB1C3 | 1 | self-making DH5α 2/23 | 10 | 11 | CP |
9/22 T7-RBS-dddD(3)-5 | 1 | self-making DH5α 2/23 | 10 | 11 | CP |
10/2
Kination
Add His to T7RBS Fw
ATOE-D
PCR
PrimeSTAR Premix/(µL) | Template #1/(ng) | Primer #2/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
50 | 400 | 2(each) | up to 100 | 100 |
#1 AT-pSB1C3(without (1))
#2 Fw:Add His to T7RBS/Rv:AT OE-D
DNA Purification
Sample |
---|
PCR product (1),(2) His-pSB1C3-AT |
PCR product(3) AT-pSB1C3 (AT Mota I) |
PCR product(4) AT-pSB1C3 (AT Mota D) |
PCR product(5) T7-RBS-His-pSB1C3 |
Restriction Enzyme Digestion
Dpn1/(µL) | 10/2 PCR AT-pSB1C3 miniprep product(3)/ (µL) | 10x Buffer(H)/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
1 | 8 | 5 | 36 | 50 |
Electrophoresis
Lane | Sample |
---|---|
4 | His pSB1C3-AT 10/2 DNA Purification |
5 | AT-pSB1C3 Mutation Deletion 10/2 DNA Purification td> |
6 | 1kbp GeneRuler |
7 | AT-pSB1C3 Mutation Insert 10/2 DNA Purification td> |
8 | T7-RBS AT His 10/2 DNA Purification |
1~3,9~12 | Blank |
10/3
Miniprep
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
9/14 AT-pSB1C3(1) | 119 | 2.00 | 2.19 |
10/1 SAMmt(Mutated)-pSB1C3(1) | 275 | 1.86 | 1.75 |
10/1 SAMmt(Mutated)-pSB1C3(2) | 227 | 1.93 | 1.90 |
10/1 T7-RBS-His(1) | 147 | 2.00 | 1.64 |
10/1 T7-RBS-His(2) | 107 | 1.94 | 1.74 |
Mutation PCR
Master Mix
PrimeSTARPremix/(µL) | Template 10/3 Miniprep AT-pSB1C3(1)/(µL) th> | Primer AT Mutation Insert Rv/(µL) | AT Mutation Insert Fw/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
12.5 | 119 | 0.5 | 0.5 | up to 25 | 25 |
Denature | Annealing | Extension |
---|---|---|
94°C | 55°C | 68°C |
10sec | 15sec | 20sec |
Denature-Annealing-Extension 35cycle
Gel Extraction
Lane | Sample |
---|---|
1 | His-OE-(1) |
2 | His-OE-(2) |
Electrophoresis
Lane | Sample |
---|---|
1 | AT-pSB1C3 Mutation insert 50.2°C |
2 | AT-pSB1C3 Mutation insert 52.8°C |
3 | AT-pSB1C3 Mutation insert 54.2°C |
4 | AT-pSB1C3 Mutation insert 55.7°C |
5 | AT-pSB1C3 Mutation insert 57.1°C |
6 | 10/2 DNA Purification His pSB1C3-AT |
7 | AT-pSB1C3 Mutation Deletion 50.2°C |
8 | AT-pSB1C3 Mutation Deletion 52.8°C |
9 | AT-pSB1C3 Mutation Deletion 54.2°C |
10 | AT-pSB1C3 Mutation Deletion 55.7°C |
11 | AT-pSB1C3 Mutation Deletion 57.1°C |
12 | 100bp ladder |
OE-PCR 1st
(1)
KOD Plus/(µL) | 10xBuffer for KOD Plus/(µL) | 2mM dNTPs/(µL) | 25mM MgSO4/(µL) | 10/2 MP J04450(pSB1C3)/(µL) | VF2/(µL) | Rv mutation primer T7-RBS-His/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|---|---|---|
1 | 5 | 5 | 3 | 1 | 1.5 | 1.5 | 32 | 50 |
(2)
KOD Plus/(µL) | 10xBuffer for KOD Plus/(µL) | 2mM dNTPs/(µL) | 25mM MgSO4/(µL) | 10/2 MP J04450(pSB1C3)/(µL) | Fw mutation primer T7-RBS-His/(µL) | Present from Santa/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|---|---|---|
1 | 5 | 5 | 3 | 1 | 1.5 | 1.5 | 32 | 50 |
Denature | Annealing | Extension | Cooling | Cooling |
---|---|---|---|---|
94°C | 98°C | 55°C | 68°C | 4°C |
2min | 10sec | 30sec | 3min | infinity |
Denature-Annealing-Extension 35cycle
OE-PCR 2nd
KOD Plus/(µL) | 10xBuffer for KOD Plus/(µL) | 2mM dNTPs/(µL) | 25mM MgSO4/(µL) | His OE-(1) 10/3 gel extraction product/ (µL) | His OE-(2) 10/3 gel extraction product/ (µL) | VF2/(µL) | Present from Santa/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|---|---|---|---|
1 | 5 | 5 | 3 | 1 | 1 | 1.5 | 1.5 | 31 | 50 |
Denature | Annealing | Extension | Cooling | Cooling |
---|---|---|---|---|
94°C | 98°C | 55°C | 68°C | 4°C |
2min | 10sec | 30sec | 3min | infinity |
Denature-Annealing-Extension 35cycle
Restriction Enzyme Digestion
Sample/(µL) | Xba1/(µL) | ApnL1/(µL) | 10XT/(µL) | BSA/(µL) | CIAP/(µL) | MilliQ/(µL) | Total/(µL) | |
---|---|---|---|---|---|---|---|---|
AT-pSB1C3(1) 10/3 Miniprep | 10 | 1 | 1 | 4 | 4 | 1 | 19 | 40 |
OE-PCR 2nd(T7 RBS His) without CIAP | 10 | 1 | 1 | 4 | 4 | - | 20 | 40 |
Ligation
A
Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) | ||||
---|---|---|---|---|---|---|---|---|---|
pSB1C3 10/3 XA | 2 | T7 RBS His 10/3 XA | 2 | 2xRapidLigBuffer | 5 | Promega T4 Ligase | 1 | - | 10 |
pSB1C3 10/3 XA | 2 | T7 RBS His 10/3 XA | - | 2xRapidLigBuffer | 5 | Promega T4 Ligase | 1 | 2 | 10 |
B
Vector/(µL) | Insert/(µL) | Buffer/(µL) | MilliQ/(µL) | Total/(µL) | |||
---|---|---|---|---|---|---|---|
pSB1C3 10/3 XA | 2 | T7 RBS His 10/3 XA | 2 | 2xRapidLigBuffer | 4 | - | 8 |
pSB1C3 10/3 XA | 1 | T7 RBS His 10/3 XA | - | 2xRapidLigBuffer | 4 | 1 | 6 |
Transformation
Sample | CompetentCells | Medium |
---|---|---|
10/3 Ligation products | DH5α | CP |
DNA Purification
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
10/3 OE-PCR 2nd | 204.5 | 1.82 | 2.33 |
DNA Purification
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
10/3 digestion product(1) | 29.6 | 1.53 | 1.11 |
10/3 digestion product(2) | 23.2 | 1.67 | 0.68 |
DNA Purification
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
RFP OE-1 | 33.4 | 1.58 | 1.08 |
RFP OE-2 | 51.7 | 1.70 | 1.19 |
DNA Purification
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
10/3 OE-PCR 2nd | 416.3 | 1.81 | 2.31 |
Transformation
Sample | CompetentCells |
---|---|
10/4 Ligation product | DH5α |
10/4
Gel Extraction
Sample |
---|
10/4 Digestion product |
Ligation
Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) | ||||
---|---|---|---|---|---|---|---|---|---|
pSB1C3 10/4 XA | 2 | RFP His 10/4 XA | 2 | 2x Rapid Ligation Buffer | 5 | Promega T4 Ligase | 1 | - | 10 |
pSB1C3 10/4 XA | 3 | RFP His 10/4 XA | 1 | 2x Rapid Ligation Buffer | 5 | Promega T4 Ligase | 1 | - | 10 |
pSB1C3 10/4 XA | 1 | RFP His 10/4 XA | 3 | 2x Rapid Ligation Buffer | 5 | Promega T4 Ligase | 1 | - | 10 |
pSB1C3 10/4 XA | 1 | RFP His 10/4 XA | 1 | 2x Rapid Ligation Buffer | 5 | Promega T4 Ligase | 1 | 2 | 10 |
10/5
OE-PCR 2nd
KOD Plus/(µL) | 10xBuffer for KOD Plus/(µL) | 2mM dNTPs/(µL) | 25mM MgSO4/(µL) | Primers #1/(µL) | Templates #2/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|---|---|
2 | 10 | 10 | 6 | 3(each) | 2(each) | 62 | 100 |
1. #1 VF2/Present from Santa #2 RFP OE(1)/RFP OE(2)
2. #1 VF2/Present from Santa #2 RFP OE(1)/His OE(2)
Denature | Annealing | Extension | Final Extension |
---|---|---|---|
94°C | 98°C | 55°C | 68°C |
2min | 10sec | 30sec | 100min |
Denature-Annealing-Extension 35cycle
Restriction Enzyme Digestion
Sample/(µL) | Xba1/(µL) | Spe1/(µL) | CIAP/(µL) | 10XT/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | |
---|---|---|---|---|---|---|---|---|
10/5 RPFHis(1) 10/5 OE2(1) | 5 | 1 | 1 | - | 4 | 4 | 20 | 40 |
10/5 RPFHis(2) 10/5 OE2(2) | 5 | 1 | 1 | - | 4 | 4 | 20 | 40 |
10/5 RPFHis(3) 10/4 OE2 RPF His | 5 | 1 | 1 | - | 4 | 4 | 20 | 40 |
10/5 pSB1C3 XA (1) 9/18 Mutated Bn-pSB1C310-3 | 5 | 1 | 1 | 1 | 4 | 4 | 19 | 40 |
10/5 pSB1C3 XA (2) 9/14 Mutated dddD(mutated)-pSB1C3(2) | 5 | 1 | 1 | 1 | 4 | 4 | 19 | 40 |
Gel Extraction
Lane | Sample |
---|---|
(1)-1,(2)-6 | SKY |
(1)-2,3 | 12FP His 10/5 XA(1) |
(1)-4,5 | pSB1C3 10/5 XA(2) |
(1)-6 | RFP His 10/5 XA(2) |
(2)-1 | RFP His 10/5 XA(2) |
(2)-2,3 | RFP His 10/5 XA(3) |
(2)-4,5 | pSB1C3 10/5 XA(1) |
DNA Purification
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
10/5 RFP His1 | 205.9 | 1.87 | 2.39 |
10/5 RFP His2 | 157.6 | 1.84 | 2.34 |
Ligation
Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) | ||||
---|---|---|---|---|---|---|---|---|---|
pSB1C3 10/4 XA | 1 | RFP His 10/4 XA | 1 | TOYOBO Ligation High | 2 | - | - | - | 4 |
- | - | RFP His 10/4 XA | 1 | TOYOBO Ligation High | 2 | - | - | 1 | 4 |
Transformation
Sample | CompetentCells |
---|---|
10/5 Ligation product | DH5α |
Liquid Culture
Sample |
---|
10/4 C1 |
10/4 C2 |
Gel extraction
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
RFP His XA1 | 169.8 | 1.85 | 1.13 |
RFP His XA3 | 62.6 | 1.79 | 1.11 |
pSB1C3 XA 2x | 26.0 | 1.71 | 1.01 |
10/6
Miniprep
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
10/5 Pcon RPF-His A | 133 | 1.93 | 2.24 |
10/5 Pcon RPF-His B | 133 | 1.90 | 1.83 |
10/5 Pcon RPF-His C | 137 | 1.80 | 1.72 |
10/5 Pcon RPF-His D | 142 | 1.98 | 2.61 |
PCR
10/6 Pcon RPFHisA VF
Template/(µL) | Bigdye/(µL) | 5xBuffer/(µL) | Primer/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
2.9 | 0.5 | 1.75 | 0.5 | 4.85 | 10.5 |
10/6 Pcon RPFHisA VR
Template/(µL) | Bigdye/(µL) | 5xBuffer/(µL) | Primer/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
2.9 | 0.5 | 1.75 | 0.5 | 4.85 | 10.5 |
10/6 Pcon RPFHisB VF
Template/(µL) | Bigdye/(µL) | 5xBuffer/(µL) | Primer/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
3.0 | 0.5 | 1.75 | 0.5 | 4.75 | 10.5 |
10/6 Pcon RPFHisB VR
Template/(µL) | Bigdye/(µL) | 5xBuffer/(µL) | Primer/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
3.0 | 0.5 | 1.75 | 0.5 | 4.75 | 10.5 |
10/6 Pcon RPFHisC VF
Template/(µL) | Bigdye/(µL) | 5xBuffer/(µL) | Primer/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
2.9 | 0.5 | 1.75 | 0.5 | 4.85 | 10.5 |
10/6 Pcon RPFHisC VR
Template/(µL) | Bigdye/(µL) | 5xBuffer/(µL) | Primer/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
2.9 | 0.5 | 1.75 | 0.5 | 4.85 | 10.5 |
10/6 Pcon RPFHisD VF
Template/(µL) | Bigdye/(µL) | 5xBuffer/(µL) | Primer/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
2.8 | 0.5 | 1.75 | 0.5 | 4.95 | 10.5 |
10/6 Pcon RPFHisD VR
Template/(µL) | Bigdye/(µL) | 5xBuffer/(µL) | Primer/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
2.8 | 0.5 | 1.75 | 0.5 | 4.95 | 10.5 |
Predenature | Denature | Annealing | Extension | Cooling | Cooling |
---|---|---|---|---|---|
96°C | 96°C | 50°C | 60°C | 4°C | 4°C |
2min | 10sec | 5sec | 4min | 1min | infinity |
Denature-Annealing-Extension 30cycle
10/7
PCR
AT(1)
Template/(µL) | Bigdye/(µL) | 5xBuffer/(µL) | Primer/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
4.0 | 0.5 | 1.75 | 0.5 | 3.25 | 10.5 |
AT(2)
Template/(µL) | Bigdye/(µL) | 5xBuffer/(µL) | Primer/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
2.0 | 0.5 | 1.75 | 0.5 | 5.25 | 10.5 |
AT(3)
Template/(µL) | Bigdye/(µL) | 5xBuffer/(µL) | Primer/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
2.0 | 0.5 | 1.75 | 0.5 | 5.25 | 10.5 |
AT(4)
Template/(µL) | Bigdye/(µL) | 5xBuffer/(µL) | Primer/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
2.5 | 0.5 | 1.75 | 0.5 | 4.75 | 10.5 |
Predenature | Denature | Annealing | Extension | Cooling | Cooling |
---|---|---|---|---|---|
96°C | 96°C | 50°C | 60°C | 4°C | 4°C |
2min | 10sec | 5sec | 4min | 1min | infinity |
Denature-Annealing-Extension 30cycle
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|
10/6 T7-RBS-His(2) | 12 | - | - | 1 | 1 | B | 4 | 0.4 | 21.6 | 40 |
10/6 T7-RBS-His(2) control | 1 | - | - | - | - | B | 1 | - | 8 | 10 |
10/6 T7-RBS-His(3) | 20 | - | - | 2 | 2 | B | 8 | 0.8 | 47.2 | 80 |
10/6 T7-RBS-His(3) control | 1 | - | - | - | - | B | 1 | - | 8 | 10 |
Gel Extraction
Lane | Sample |
---|---|
1 | T7RBSHis(3) 10/7 S,P control |
2 | T7RBSHis(3) 10/7 S,P Experimental |
3 | 100bp ladder |
4 | Blank |
5 | 7RBSHis(2) 10/7 S,P Experimental |
6 | 7RBSHis(2) 10/7 S,P control |
Lane | Sample |
---|---|
3 | T7RBSHis(3) 10/7 S,P Experimental |
1,2,4,5,6 | Blank |
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
T7RBSHis(3) low | 38 | 1.52 | 1.30 |
T7RBSHis(2) | 46 | 1.49 | 1.53 |
T7RBSHis(3) high | 28 | 2.04 | 0.38 |
10/8
Liquid Culture
Sample | Medium |
---|---|
BL21 | LB |
Liquid Culture
Sample | Medium |
---|---|
9/30 AT-pSB1C3 insertion mutated | CP |
9/30 SAMmt-pSB1C3 mutated | CP |
10/10
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|
AT(Mutation Insert)-pSB1C3(2) | 20 | - | 1 | - | 1 | D | 4 | 0.4 | 3.6 | 40 |
Dephosphorylation
10/8 T7 RBSHis(2)S,P/(µL) | CIAP/(µL) | 10xBuffer/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
12 | 1 | 2 | 5 | 20 |
37°C | 50°C | 15°C |
30min | 15min | 30min |
Miniprep
Sample |
---|
Liquid Culture product SAM(Mutated)-pSB1C3(1)~(4) |
Cloning PCR
2X KAPA HiFI HotStart ReadyMix/(µL) | AT OE-A/(µL) | AT cloning for His/(µL) | 10/7 M AT(Mutation Insert)-pSB1C3(2)/(µL) th> | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
12.5 | 0.75 | 0.75 | up to 25 | 11 | 25 |
Predenature | Denature | Annealing | Extension | Final Extension |
---|---|---|---|---|
95°C | 98°C | 60°C | 72°C | 72°C |
5min | 20sec | 15sec | 90sec | 5sec |
Denature-Annealing-Extension 35cycle
Gel Extraction
Lane | Sample |
---|---|
1 | AT pSB1C3 (Mutation Insert)(2)10/3 |
2 | Blank |
3 | 100bp ladder |
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|
10/10 Miniprep product SAMmt(Mutated)-pSB1C3(1) | 10 | - | - | - | 1 | E | 4 | 0.4 | 24.6 | 40 |
10/10 Miniprep product SAMmt(Mutated)-pSB1C3(2) | 10 | - | - | - | 1 | E | 4 | 0.4 | 24.6 | 40 |
10/10 Miniprep product SAMmt(Mutated)-pSB1C3(3) | 10 | - | - | - | 1 | E | 4 | 0.4 | 24.6 | 40 |
10/10 Miniprep product SAMmt(Mutated)-pSB1C3(4) | 10 | - | - | - | 1 | E | 4 | 0.4 | 24.6 | 40 |
Ligation
Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) | ||||
---|---|---|---|---|---|---|---|---|---|
T7 RBS-pSB1C3 S,P | 2 | 10/10 gel extraction product(true)XP | 2 | 2xRapidLigaseBuffer | 5 | Promega T4 Ligase | 1 | - | 10 |
T7 RBS-pSB1C3 S,P | 1 | 10/10 gel extraction product(true)XP | 3 | 2xRapidLigaseBuffer | 5 | Promega T4 Ligase | 1 | - | 10 |
T7 RBS-pSB1C3 S,P | 2 | 10/10 gel extraction product(true)XP | 0 | 2xRapidLigaseBuffer | 5 | Promega T4 Ligase | 1 | 2 | 10 |
T7 RBS-pSB1C3 S,P | 2 | 10/10 gel extraction product(true)XP | 2 | 2xRapidLigaseBuffer | 5 | Promega T4 Ligase | 1 | - | 10 |
T7 RBS-pSB1C3 S,P | 1 | 10/10 gel extraction product(true)XP | 3 | 2xRapidLigaseBuffer | 5 | Promega T4 Ligase | 1 | - | 10 |
T7 RBS-pSB1C3 S,P | 2 | 10/10 gel extraction product(true)XP | 0 | 2xRapidLigaseBuffer | 5 | Promega T4 Ligase | 1 | 2 | 10 |
Transformation
Sample | CompetentCells | Medium |
---|---|---|
10/10 Ligation product | BL21(DE3) | CP |
10/11
Miniprep
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
T7RBSHis(2) | 217.4 | 1.92 | 2.16 |
T7RBSHis(3) | 418.0 | 1.95 | 2.30 |
AT-pSB1C3(2) | 258.0 | 1.96 | 2.25 |
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | CIAP/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|---|
10/11 miniprep product T7RBSHis(2) | 10 | - | - | 2 | 2 | B | 8 | 0.8 | 47.2 | 80 | |
10/11 miniprep product T7RBSHis(3) | 10 | - | - | 2 | 2 | B | 8 | 0.8 | 57.2 | 80 | |
10/11 miniprep product AT-pSB1C3(2) | 10 | - | 1 | - | 1 | B | 4 | 0.4 | 0 | 23.6 | 40 |
10/10 miniprep product SAM(mutated)-pSB1C3-(1) td> | 10 | - | - | 1 | - | B | 4 | 0.4 | 0 | 24.6 | 40 |
10/10 miniprep product SAM(mutated)-pSB1C3-(2) td> | 10 | - | - | 1 | - | B | 4 | 0.4 | 0 | 24.6 | 40 |
10/10 miniprep product SAM(mutated)-pSB1C3-(3) td> | 10 | - | - | 1 | - | B | 4 | 0.4 | 0 | 24.6 | 40 |
10/10 miniprep product SAM(mutated)-pSB1C3-(4) td> | 10 | - | - | 1 | - | B | 4 | 0.4 | 0 | 24.6 | 40 |
DNA Purification
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
10/10 PCR Cloning product AT | 207.6 | 1.88 | 2.00 |
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|
10/11 DNA Purification product AT(His) | 10 | 0 | 1 | 0 | 1 | D | 4 | 0.4 | 24.6 | 40 |
Transformation
Sample | CompetentCells | Medium |
---|---|---|
10/10 Ligation | BL21(DE3) | CP |
10/13
Miniprep
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
1. 10/13 T7-RBS (1) | 334.6 | 1.91 | 2.15 |
2. 10/13 T7-RBS (1) | 319.6 | 1.87 | 1.72 |
Electrophoresis
Lane | Sample | ||
---|---|---|---|
1 | - | ||
2 | - | ||
3 | - | ||
4 | 1kb ladder | ||
5 | 10/11 SAMmt-pSB1C3 (S)-(2) | ||
6 | 10/11 SAMmt-pSB1C3 (S)-(3) | ||
7 | 10/11 SAMmt-pSB1C3 (S)-(1) | ||
8 | 10/11 SAMmt-pSB1C3 (S)-(4) | ||
9 | 1kb ladder |
Gel Extraction
Lane | Sample |
---|---|
1 | - |
2 | - |
3 | 10/11 AT-pSB1C3(X,P)-(2) |
4 | - |
5 | - |
6 | 10/11 AT-His(X,P) |
7 | - |
8 | 1kb ladder |
9 | - |
10 | 10/11 T7-RBS-His (S,P)-(2) |
11 | - |
12 | - |
13 | 10/11 T7-RBS-His (S,P)-(3) |
Miniprep
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
1. 10/13 AT (X,P)(Gel Extraction Product) | 43.1 | 1.88 | 0.77 |
2. 10/13 AT-His (X,P)(Gel Extraction Product) | 34.6 | 1.96 | 0.37 |
3. 10/13 T7-RBS-His(2) (S,P)(Gel Extraction Product) | 57.7 | 1.84 | 1.29 |
4. 10/13 T7-RBS-His(3) (S,P)(Gel Extraction Product) | 37.5 | 1.81 | 1.10 |
Ligation
Vector/(µL) | Insert/(µL) | Ligation high/(µL) | Total/(µL) | ||
---|---|---|---|---|---|
1. 9/19 T7-RBS(S,P)(DNA purification product) | 0.8 | 10/13 AT(X,P)(Gel Extraction Product) | 2.0 | 2.8 | 5.6 |
2. 10/13 T7-RBS-His(S,P)-(2) (Gel Extraction Product) | 0.7 | 10/13 AT-His(X,P)(Gel Extraction Product) | 2.5 | 3.2 | 6.4 |
3. 10/13 T7-RBS-His(S,P)-(3) (Gel Extraction Product) | 0.8 | 10/13 AT-His(X,P)(Gel Extraction Product) | 1.9 | 2.7 | 5.4 |
4. 9/19 T7-RBS(S,P)(DNA purification product) | 0.8 | MilliQ | 2.0 | 2.8 | 5.6 |
5. 10/13 T7-RBS-His(S,P)-(2) (Gel Extraction Product) | 0.7 | MilliQ | 2.5 | 3.2 | 6.4 |
6. 10/13 T7-RBS-His(S,P)-(3) (Gel Extraction Product) | 0.8 | MilliQ | 1.9 | 2.7 | 5.4 |
Transformation
Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium | ||
---|---|---|---|---|---|
10/13 Ligation Product (from 1 to 6) | 2 | DH5α | 10 | 12 | CP |
T7-RBS (1)(Miniprep product) | 2 | BL21(DE3) | 10 | 12 | CP |
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|
10/13 T7-RBS (1) | 20 | - | - | 3 | 3 | B | 12 | 1.2 | 79.8 | 120 |
Cloning
2x KAPA HiFi HotStart ReadyMix/(µL) | Template DNA #1/(µL) | Primers #2/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|
12.5 | pick up | 0.75 | 11 | 25 |
(1)
#1: 10/10 SAMmt-pSB1C3(3) (After mutation, miniprep product)
#2: VF2, Rv SAMmt-His
(2)
#1: 10/10 SAMmt-pSB1C3(3) (After mutation, miniprep product)
#2: SAMmt(OE-A), Rv SAMmt-His
Predenature | Denature | Annealing | Extension | Last Extension | Cooling |
---|---|---|---|---|---|
95°C | 98°C | 60°C | 72°C | 72°C | 12°C |
5min | 20sec | 15sec | 1min15sec | 5min | Infinity |
Denature-Annealing-Extension 30cycle
DNA Purification
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
1. 10/14 SAMmt-pSB1C3(3)(PCR product) | 206.7 | 1.88 | 11.17 |
2. 10/14 SAMmt-pSB1C3(3)(PCR product) | 187.9 | 1.89 | 28.58 |
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|
10/11 AT-pSB1C3 (2)(Miniprep product) | 5 | - | 0.5 | - | 0.5 | D | 2 | 0.2 | 11.8 | 20 |
10/14 SAMmt-His (1)(DNA purification product) | 10 | - | 1 | - | 1 | D | 2 | 0.4 | 23.6 | 40 |
10/14 SAMmt-His (2)(DNA purification product) | 10 | - | 1 | - | 1 | D | 2 | 0.4 | 23.6 | 40 |
10/14
Gel Extraction
(1)
Lane | Sample |
---|---|
1 | 1kb ladder |
2 | - |
3 | 10/14 AT(X,P) |
4 | - |
5 | - |
6 | 10/14 SAMmt-His(2)(X,P) |
(2)
Lane | Sample |
---|---|
1 | - |
2 | - |
3 | 10/13 T7-RBS(S,P) |
4 | 1kb ladder |
5 | - |
6 | 10/11 T7-RBS-His(3) (S,P) |
Transformation
Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium | |||||||
---|---|---|---|---|---|---|---|---|---|---|
10/13 Ligation product (from 1 to 6) | 2 | DH5α | 10 | 12 | CP | |||||
10/13 T7-RBS(1) | 2 | BL21(DE3) | 10 | 12 | CP | |||||
Control (pBR322) | 2 | DH5α | 10 | 12 | Amp | |||||
Control (pBR322) | 2 | DH5α | 10 | 12 | Amp | |||||
Control (pBR322) | 2 | BL21(DE3) | 10 | 12 | Amp |
2x KAPA HiFi HotStart ReadyMix/(µL) | 9/22 T7-RBS-dddD(After mutation, miniprep product)(3)/(µL) | VF2/(µL) | Rv dddD cloning for His/(µL) | MilliQ/(µL) | Total/(µL) |
---|---|---|---|---|---|
12.5 | pick up | 0.75 | 0.75 | 11 | 25 |
Predenature | Denature | Annealing | Extension | Last Extension | Cooling |
---|---|---|---|---|---|
95°C | 98°C | 60°C | 72°C | 72°C | 12°C |
5min | 20sec | 15sec | 1min15sec | 5min | Infinity |
Denature-Annealing-Extension 30cycle
DNA Purification
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
10/14 AT(X,P)(Gel Extraction product) | 32 | 1.82 | 1.21 |
10/14 SAMmt-His(2)(X,P)(Gel Extraction product) | 70 | 1.78 | 1.01 |
10/14 T7-RBS(S,P)(Gel Extraction product) | 88 | 1.66 | 2.04 |
10/14 T7-RBS-His(3) (S,P)(Gel Extraction product) | 156 | 1.54 | 1.10 |
10/15 dddD(After mutation, PCR product) | 346 | 1.85 | 1.44 |
Restriction Enzyme Digestion
Sample/(µL) | EcoR1/(µL) | Xba1/(µL) | Spe1/(µL) | Pst1/(µL) | Buffer/(µL) | BSA/(µL) | MilliQ/(µL) | Total/(µL) | ||
---|---|---|---|---|---|---|---|---|---|---|
10/15 dddD(After mutation, DNA purification product) | 5.8 | - | 1 | - | 1 | D | 4 | 0.4 | 27.8 | 40 |
Liquid Culture
Sample | Medium |
---|---|
9/8 T'-RBS-pSB1C3(1) | CP |
REDOX-pSB1C3 | CP |
10/3 AT-pSB1C3 | CP |
T7-RBS-His(2) | CP |
10/16
Gel Extraction/Electrophoresis
Lane | Sample |
---|---|
1 | 1kbpGeneRuler |
2 | dddD(His)X,P |
Miniprep
Sample | Concentration/(µg/mL) | 260/280 | 260/230 |
---|---|---|---|
T7RBS-pSB1C3 (1) 10/16 Miniprep product | 471 | 1.90 | 1.85 |
T7RBS-pSB1C3 (2) 10/16 Miniprep product A | 380 | 1.92 | 2.03 |
T7RBS-pSB1C3 (2) 10/16 Miniprep product B | 372 | 1.92 | 1.64 |
AC-pSB1C3(2) 10/16 Miniprep product | 473 | 1.90 | 1.68 |
REDOX-pSB1C3 10/16 Miniprep product | 530 | 1.93 | 1.81 |
dddD(His)X,P 10/16 gel extraction product | 55 | 1.40 | 1.36 |
QpSB1C3 10/16 Miniprep product | 276 | 1.86 | 2.01 |
PLQ-4A5 10/16 Miniprep product | 370 | 1.87 | 2.07 |
Ligation
Toyobo Ligationhigh
Vector/(µL) | Insert/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) | |||
---|---|---|---|---|---|---|---|
10/14 T7-RBS S,P | 2 | 10/14 AT X,P | 2 | 2xligationhigh | 4 | - | 8 |
10/14 T7-RBS S,P | 2 | 10/14 AT X,P | - | 2xligationhigh | 4 | 2 | 8 |
10/14 T7-RBS-His S,P | 1 | 10/13 AT-His X,P | 2 | 2xligationhigh | 4 | 1 | 8 |
10/14 T7-RBS-His S,P | 1 | 10/13 AT-His X,P | - | 2xligationhigh | 4 | 3 | 8 |
Promega
Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) | ||||
---|---|---|---|---|---|---|---|---|---|
10/14 T7-RBS S,P | 2 | 10/14 AT X,P | 2 | 2xRapidLigationBuffer | 5 | T4Ligase | 1 | - | 10 |
10/14 T7-RBS S,P | 2 | 10/14 AT X,P | - | 2xRapidLigationBuffer | 5 | T4Ligase | 1 | 2 | 10 |
10/14 T7-RBS-His S,P | 1 | 10/13 AT-His X,P | 2 | 2xRapidLigationBuffer | 5 | T4Ligase | 1 | 1 | 10 |
10/14 T7-RBS-His S,P | 2 | 10/14 SAM-His X,P | 2 | 2xRapidLigationBuffer | 5 | T4Ligase | 1 | - | 10 |
10/14 T7-RBS-His S,P | 2 | REDOX-His | 2 | 2xRapidLigationBuffer | 5 | T4Ligase | 1 | - | 10 |
10/14 T7-RBS-His S,P | 2 | REDOX-His | - | 2xRapidLigationBuffer | 5 | T4Ligase | 1 | 2 | 10 |
NewIngland BioLabs
Vector/(µL) | Insert/(µL) | Buffer/(µL) | Ligase/(µL) | MilliQ/(µL) | Total/(µL) | ||||
---|---|---|---|---|---|---|---|---|---|
10/14 T7-RBS-His S,P | 2 | 10/14 SAM-His X,P | 2 | 10xLigationBuffer | 0.5 | T4 Ligase | 0.5 | - | 5 |
10/14 T7-RBS-His S,P | 2 | REDOX-His | 2 | 10xLigationBuffer | 0.5 | T4 Ligase | 0.5 | - | 5 |
10/14 T7-RBS-His S,P | 2 | REDOX-His | - | 10xLigationBuffer | 0.5 | T4 Ligase | 0.5 | 2 | 5 |
Transformation
Sample/(µL) | CompetentCells/(µL) | Total/(µL) | Medium | ||
---|---|---|---|---|---|
Toyobo T7-AT | 2 | BL21 | 10 | 12 | CP |
Toyobo T7-AT control | 2 | BL21 | 10 | 12 | CP |
Toyobo T7-AT-His | 2 | BL21 | 10 | 12 | CP |
Toyobo T7-AT-His control | 2 | BL21 | 10 | 12 | CP |
10/13 Miniprep product T7-RBS(1)B | 9 | BL21 | 90 | 99 | CP |
Toyobo T7-AT | 4 | DH5α | 20 | 24 | CP |
Toyobo T7-AT control | 4 | DH5α | 20 | 24 | CP |
Toyobo T7-AT-His | 4 | DH5α | 20 | 24 | CP |
Toyobo T7-AT-His control | 4 | DH5α | 20 | 24 | CP |
Promega T7-AT | 5 | DH5α | 30 | 35 | CP |
Promega T7-AT control | 5 | DH5α | 30 | 35 | CP |
Promega T7-AT-His | 5 | DH5α | 30 | 35 | CP |
Promega T7-SAM-His | 5 | DH5α | 30 | 35 | CP |
Promega T7-REDOX-His | 5 | DH5α | 30 | 35 | CP |
Promega T7-REDOX-His control | 5 | DH5α | 30 | 35 | CP |
NewIngland T7-SAM-His | 5 | DH5α | 30 | 35 | CP |
NewIngland T7-REDOX-His | 5 | DH5α | 30 | 35 | CP |
NewIngland T7-REDOX-His control | 5 | DH5α | 30 | 35 | CP |