"
Template:Kyoto/Notebook/DMS
From 2014.igem.org
(Difference between revisions)
| (44 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 2,122: | Line 2,122: | ||
<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> | ||
| Line 2,210: | 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> | ||
| Line 2,231: | Line 2,231: | ||
<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> | ||
| Line 2,241: | Line 2,241: | ||
<h4>Cloning</h4> | <h4>Cloning</h4> | ||
| - | <span class= | + | <span class="kyoto-author">Yasuda</span> |
<table> | <table> | ||
<tr> | <tr> | ||
| Line 2,291: | Line 2,291: | ||
<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 2,298: | Line 2,298: | ||
<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> | ||
| Line 2,354: | Line 2,354: | ||
<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> | ||
| Line 2,364: | Line 2,364: | ||
<img src="https://static.igem.org/mediawiki/2014/8/8b/Kyoto-dms-0903_Gel_Extraction_No1_after.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> | ||
| Line 2,371: | Line 2,371: | ||
<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 2,385: | 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 2,407: | 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> | ||
| Line 2,424: | Line 2,424: | ||
<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 2,567: | 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> | ||
| Line 2,580: | Line 2,580: | ||
<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 2,617: | 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> | ||
| Line 2,728: | 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 2,797: | Line 2,797: | ||
<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 2,916: | 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 2,930: | Line 2,930: | ||
<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 2,942: | 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 3,009: | Line 3,009: | ||
<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 3,018: | Line 3,018: | ||
<h4>Ligation</h4> | <h4>Ligation</h4> | ||
| - | <span class= | + | <span class="kyoto-author">Li</span> |
<table> | <table> | ||
<p>(1)Sample</p> | <p>(1)Sample</p> | ||
| Line 3,029: | 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 3,038: | 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> | ||
| Line 3,046: | Line 3,046: | ||
<h4>PCR Cloning</h4> | <h4>PCR Cloning</h4> | ||
| - | <span class= | + | <span class="kyoto-author">Murata</span> |
<table> | <table> | ||
| Line 3,096: | 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 3,169: | 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 3,184: | Line 3,184: | ||
<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 3,191: | Line 3,191: | ||
<h4>Cloning PCR</h4> | <h4>Cloning PCR</h4> | ||
| - | <span class= | + | <span class="kyoto-author">Murata</span> |
<table> | <table> | ||
<tr> | <tr> | ||
| Line 3,213: | 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 3,315: | 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 3,331: | Line 3,331: | ||
<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 3,343: | Line 3,343: | ||
<h4>Overlap Extension(OE)-PCR</h4> | <h4>Overlap Extension(OE)-PCR</h4> | ||
| - | <span class= | + | <span class="kyoto-author">Murata</span> |
<table> | <table> | ||
<tr> | <tr> | ||
| Line 3,410: | Line 3,410: | ||
<h4>Genome Cloning</h4> | <h4>Genome Cloning</h4> | ||
| - | <span class= | + | <span class="kyoto-author">Yasuda</span> |
<table> | <table> | ||
<tr> | <tr> | ||
| Line 3,446: | 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 3,527: | Line 3,527: | ||
<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 3,534: | 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 3,553: | Line 3,553: | ||
<h4>OE-PCR</h4> | <h4>OE-PCR</h4> | ||
| - | <span class= | + | <span class="kyoto-author">Shimizu</span> |
<table> | <table> | ||
<tr> | <tr> | ||
| Line 3,624: | 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 3,645: | 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 3,652: | 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 3,661: | Line 3,661: | ||
<h4>OE-PCR</h4> | <h4>OE-PCR</h4> | ||
| - | <span class= | + | <span class="kyoto-author">Shimizu</span> |
<table> | <table> | ||
<tr> | <tr> | ||
| Line 3,725: | 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 3,739: | Line 3,739: | ||
<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 3,845: | 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> | ||
| Line 3,871: | Line 3,871: | ||
<tr> | <tr> | ||
<th>Prime STAR MAX Premix(2x)</th> | <th>Prime STAR MAX Premix(2x)</th> | ||
| - | <th>Fw | + | <th>Fw pSB1C3-his-F-v2 primer</th> |
| - | <th>Rv | + | <th>Rv pSB1C3 Histag mutation primer</th> |
<th>9/10 MP T7-RBS (x3000)</th> | <th>9/10 MP T7-RBS (x3000)</th> | ||
<th>MilliQ</th> | <th>MilliQ</th> | ||
| Line 3,936: | 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 3,943: | 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 4,065: | Line 4,065: | ||
<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 4,086: | 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 4,111: | Line 4,111: | ||
<h4>OE-PCR</h4> | <h4>OE-PCR</h4> | ||
| - | <span class= | + | <span class="kyoto-author">Yasuda</span> |
<table> | <table> | ||
<tr> | <tr> | ||
| Line 4,186: | 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> | ||
| Line 4,199: | Line 4,199: | ||
<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 4,210: | 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> | ||
| Line 4,220: | Line 4,220: | ||
<h4>Sequence</h4> | <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 4,255: | Line 4,255: | ||
<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> | ||
| Line 4,264: | 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 4,357: | 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 4,371: | Line 4,371: | ||
<h4>Cloning PCR (for Histag)</h4> | <h4>Cloning PCR (for Histag)</h4> | ||
| - | <span class= | + | <span class="kyoto-author">Murata</span> |
<table> | <table> | ||
<tr> | <tr> | ||
| Line 4,423: | 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 4,441: | 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 4,450: | 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 4,567: | 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 4,580: | 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> | ||
| Line 4,588: | Line 4,588: | ||
<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>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 4,594: | 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 4,606: | 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 4,629: | Line 4,629: | ||
<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 4,637: | 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 4,696: | Line 4,696: | ||
<h4>Liquid Culture</h4> | <h4>Liquid Culture</h4> | ||
| - | <span class= | + | <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>Amp</td></tr> | <tr><td>9/11 SAMmt-(3)</td><td>Amp</td></tr> | ||
<tr><td>9/8 T7-RBS-(1)</td><td>CP</td></tr> | <tr><td>9/8 T7-RBS-(1)</td><td>CP</td></tr> | ||
| Line 4,783: | 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 4,796: | 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 4,802: | 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/cc/Kyoto-dms-0913_Gel_Extention_No1_before.jpg"> | ||
| Line 4,826: | Line 4,826: | ||
<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 4,836: | 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 4,880: | Line 4,880: | ||
<h4>Colony PCR</h4> | <h4>Colony PCR</h4> | ||
| - | <span class= | + | <span class="kyoto-author">Yasuda</span> |
| Line 4,935: | 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 4,942: | 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> | ||
| Line 4,949: | Line 4,949: | ||
<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 5,053: | Line 5,053: | ||
<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 5,069: | 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 5,091: | 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 5,105: | 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 5,124: | 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> | ||
| Line 5,171: | 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 5,178: | 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 5,191: | 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 5,214: | 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 5,236: | Line 5,236: | ||
<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>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>9/11 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 5,295: | 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> | ||
| Line 5,315: | Line 5,315: | ||
<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 5,324: | 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 5,343: | 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 5,356: | 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 5,381: | 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 5,400: | 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 5,420: | Line 5,420: | ||
<h4>Transformation</h4> | <h4>Transformation</h4> | ||
| - | <span class= | + | <span class="kyoto-author">Murata</span> |
<table> | <table> | ||
| Line 5,435: | 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 5,451: | 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> | ||
| Line 5,459: | Line 5,459: | ||
<tr><td>DiDECARB-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>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 5,491: | 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 5,500: | Line 5,500: | ||
<span class="kyoto-author">Tatsui and Nakamura</span> | <span class="kyoto-author">Tatsui and Nakamura</span> | ||
| - | <p>9/14 dddD- | + | <p>9/14 dddD-pSB1C3(Miniprep product)-(1)<br> |
(1)dddD sequence VF2</p> | (1)dddD sequence VF2</p> | ||
<table> | <table> | ||
| Line 5,680: | Line 5,680: | ||
| - | <p>9/14 dddD- | + | <p>9/14 dddD-pSB1C3(Miniprep product)-(2)</p> |
<p>(1)dddD sequence VF2</p> | <p>(1)dddD sequence VF2</p> | ||
<table> | <table> | ||
| Line 5,827: | Line 5,827: | ||
| - | <p>9/15 AT- | + | <p>9/15 AT-pSB1C3(Miniprep product)-(1)</p> |
<p>(1)VF</p> | <p>(1)VF</p> | ||
<table> | <table> | ||
| Line 5,904: | Line 5,904: | ||
<tr><td>1.*(written in magnetosome)</td><td>*</td></tr> | <tr><td>1.*(written in magnetosome)</td><td>*</td></tr> | ||
<tr><td>2.*</td><td>*</td></tr> | <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 5,989: | 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/2014/9/92/Kyoto-dms-0916_Gel_Extention_No1_before.jpg"> | ||
| Line 5,999: | Line 5,999: | ||
<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/2014/3/32/Kyoto-dms-0916_Gel_Extention_No2_before.jpg"> | ||
| Line 6,014: | Line 6,014: | ||
<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> | ||
| Line 6,025: | 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> | ||
| Line 6,035: | Line 6,035: | ||
<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 6,043: | 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 6,055: | 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 6,070: | 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 6,081: | 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 6,101: | 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 6,190: | 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 6,201: | 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 6,213: | 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 6,263: | Line 6,263: | ||
<br> | <br> | ||
#1: VF2<br> | #1: VF2<br> | ||
| - | #2: 9/14 dddD( | + | #2: 9/14 dddD(After mutation)(miniprep product) 0.25µL<br> |
<br> | <br> | ||
#1: VR<br> | #1: VR<br> | ||
| - | #2: 9/14 dddD( | + | #2: 9/14 dddD(After mutation)(miniprep product) 0.25µL<br> |
<br> | <br> | ||
<br>(3) | <br>(3) | ||
<br> | <br> | ||
#1: VF2<br> | #1: VF2<br> | ||
| - | #2: 9/14 T7-RBS-His- | + | #2: 9/14 T7-RBS-His-pSB1C3 (miniprep product) 0.34µL<br> |
<br> | <br> | ||
#1: Sequence No.5<br> | #1: Sequence No.5<br> | ||
| - | #2: 9/14 T7-RBS-His- | + | #2: 9/14 T7-RBS-His-pSB1C3 (miniprep product) 0.34µL<br> |
<br> | <br> | ||
<br>(4) | <br>(4) | ||
<br> | <br> | ||
#1: VR<br> | #1: VR<br> | ||
| - | #2: 9/16 AT- | + | #2: 9/16 AT-pSB1C3-(1) (miniprep product) 0.5µL<br> |
<br> | <br> | ||
#1: VF2<br> | #1: VF2<br> | ||
| - | #2: 9/16 AT- | + | #2: 9/16 AT-pSB1C3-(1) (miniprep product) 0.5µL<br> |
<br> | <br> | ||
#1: Sequence No.1<br> | #1: Sequence No.1<br> | ||
| - | #2: 9/16 AT- | + | #2: 9/16 AT-pSB1C3-(1) (miniprep product) 0.5µL<br> |
<br> | <br> | ||
#1: Secuence No.2<br> | #1: Secuence No.2<br> | ||
| - | #2: 9/16 AT- | + | #2: 9/16 AT-pSB1C3-(1) (miniprep product) 0.5µL<br> |
</P> | </P> | ||
| Line 6,332: | Line 6,332: | ||
<tr><td>2</td><td>Control</td></tr> | <tr><td>2</td><td>Control</td></tr> | ||
<tr><td>3</td><td>-</td></tr> | <tr><td>3</td><td>-</td></tr> | ||
| - | <tr><td>4</td><td>9/16 AT- | + | <tr><td>4</td><td>9/16 AT-pSB1C3(X,P)</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> | ||
| Line 6,492: | Line 6,492: | ||
<tr> | <tr> | ||
<td>8-9</td> | <td>8-9</td> | ||
| - | <td>9/16 dddD | + | <td>9/16 dddD-T7-RBS (After mutation)(1)-(2)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>10-16</td> | <td>10-16</td> | ||
| - | <td>9/16 SAMmt- | + | <td>9/16 SAMmt-pSB1C3 (1)-(7)</td> |
</tr> | </tr> | ||
| Line 6,524: | Line 6,524: | ||
<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>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 | + | <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> | <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> | ||
| Line 6,539: | Line 6,539: | ||
<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 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 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- | + | <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> | <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> | </table> | ||
| Line 6,556: | Line 6,556: | ||
<tr><td>1-2</td><td>9/15 AT-His-T7-RBS His (1)(2)</td></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>3-7</td><td>9/15 AT-His-T7-RBS His (1)-(5)</td></tr> | ||
| - | <tr><td>8</td><td>9/16 dddD | + | <tr><td>8</td><td>9/16 dddD-T7-RBS(After mutation)(1)</td></tr> |
<tr><td>9</td><td>1kb ladder</td></tr> | <tr><td>9</td><td>1kb ladder</td></tr> | ||
</table> | </table> | ||
| Line 6,565: | Line 6,565: | ||
<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</td><td>9/16 dddD | + | <tr><td>2</td><td>9/16 dddD-T7-RBS(After mutation)(2)</td></tr> |
| - | <tr><td>3-8</td><td>9/16 SAMmt- | + | <tr><td>3-8</td><td>9/16 SAMmt-pSB1C3 (1)-(6)</td></tr> |
</table> | </table> | ||
<img src="https://static.igem.org/mediawiki/2014/9/9c/Kyoto-dms-0917_Elecrophoresis_No2.jpg"> | <img src="https://static.igem.org/mediawiki/2014/9/9c/Kyoto-dms-0917_Elecrophoresis_No2.jpg"> | ||
| Line 6,574: | Line 6,574: | ||
<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</td><td>9/16 SAMmt- | + | <tr><td>2</td><td>9/16 SAMmt-pSB1C3 (7)</td></tr> |
</table> | </table> | ||
| Line 6,584: | Line 6,584: | ||
<tr><th>Sample</th></tr> | <tr><th>Sample</th></tr> | ||
<tr><td>9/16 AT His-T7-RBS His (2)-(4)</td></tr> | <tr><td>9/16 AT His-T7-RBS His (2)-(4)</td></tr> | ||
| - | <tr><td>9/16 SAMmt- | + | <tr><td>9/16 SAMmt-pSB1C3 (2)(4)</td></tr> |
</table> | </table> | ||
| Line 6,615: | Line 6,615: | ||
<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 (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 T7-AT-His (4)</td><td>145</td><td>1.83</td><td>2.16</td></tr> | ||
| - | <tr><td>9/16 SAMmt- | + | <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- | + | <tr><td>9/16 SAMmt-pSB1C3 (4)</td><td>190</td><td>1.75</td><td>2.12</td></tr> |
</table> | </table> | ||
| Line 6,731: | Line 6,731: | ||
<h4>Liquid Culture</h4> | <h4>Liquid Culture</h4> | ||
| - | <span class= | + | <span class="kyoto-author">Honda</span> |
<table> | <table> | ||
<tr> | <tr> | ||
| Line 6,745: | Line 6,745: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>9/14 AT- | + | <td>9/14 AT-pSB1C3-(1)</td> |
<td>CP</td> | <td>CP</td> | ||
</tr> | </tr> | ||
| Line 6,821: | Line 6,821: | ||
<h4>UV-spequtrum measurement</h4> | <h4>UV-spequtrum measurement</h4> | ||
| - | <span class= | + | <span class="kyoto-author">Yasuda and Murata</span> |
Ethanenitrile solution of saturated MTOB (x10) | Ethanenitrile solution of saturated MTOB (x10) | ||
| Line 6,829: | Line 6,829: | ||
<h4>Mutation PCR</h4> | <h4>Mutation PCR</h4> | ||
| - | <span class= | + | <span class="kyoto-author">Honda</span> |
<p>SAMmt (SpeI)</p> | <p>SAMmt (SpeI)</p> | ||
<table> | <table> | ||
<tr> | <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> | ||
<tr> | <tr> | ||
| Line 6,885: | Line 6,885: | ||
<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>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>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- | + | <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>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- | + | <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</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>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- | + | <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> | </table> | ||
| Line 6,899: | Line 6,899: | ||
<table> | <table> | ||
<tr> | <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> | ||
<tr> | <tr> | ||
| Line 6,943: | Line 6,943: | ||
<td>50-65°C</td> | <td>50-65°C</td> | ||
<td>68°C</td> | <td>68°C</td> | ||
| - | < | + | <td>15°C</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 6,980: | Line 6,980: | ||
<td>50-65°C</td> | <td>50-65°C</td> | ||
<td>68°C</td> | <td>68°C</td> | ||
| - | < | + | <td>15°C</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 7,017: | Line 7,017: | ||
<td>50-65°C</td> | <td>50-65°C</td> | ||
<td>68°C</td> | <td>68°C</td> | ||
| - | < | + | <td>15°C</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 7,054: | Line 7,054: | ||
<td>50-65°C</td> | <td>50-65°C</td> | ||
<td>68°C</td> | <td>68°C</td> | ||
| - | < | + | <td>15°C</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 7,087: | Line 7,087: | ||
<td>50-65°C</td> | <td>50-65°C</td> | ||
<td>68°C</td> | <td>68°C</td> | ||
| - | < | + | <td>15°C</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 7,124: | Line 7,124: | ||
<td>50-65°C</td> | <td>50-65°C</td> | ||
<td>68°C</td> | <td>68°C</td> | ||
| - | < | + | <td>15°C</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 7,162: | Line 7,162: | ||
<td>50-65°C</td> | <td>50-65°C</td> | ||
<td>68°C</td> | <td>68°C</td> | ||
| - | < | + | <td>15°C</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 7,237: | Line 7,237: | ||
<tr> | <tr> | ||
<td>3</td> | <td>3</td> | ||
| - | <td>SAM- | + | <td>SAM-pSB1C3(4)</td> |
<td>2.8</td> | <td>2.8</td> | ||
<td>0.5</td> | <td>0.5</td> | ||
| Line 7,270: | Line 7,270: | ||
<tr> | <tr> | ||
<td>6</td> | <td>6</td> | ||
| - | <td>SAM- | + | <td>SAM-pSB1C3(4)</td> |
<td>2.8</td> | <td>2.8</td> | ||
<td>0.5</td> | <td>0.5</td> | ||
| Line 7,303: | Line 7,303: | ||
<tr> | <tr> | ||
<td>9</td> | <td>9</td> | ||
| - | <td>SAM- | + | <td>SAM-pSB1C3(4)</td> |
<td>2.8</td> | <td>2.8</td> | ||
<td>0.5</td> | <td>0.5</td> | ||
| Line 7,375: | Line 7,375: | ||
<tr> | <tr> | ||
<td>3</td> | <td>3</td> | ||
| - | <td>T7-RBS-AT-His- | + | <td>T7-RBS-AT-His-pSB1C3 S,P</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>6</td> | <td>6</td> | ||
| - | <td>T7-RBS- | + | <td>T7-RBS-pSB1C3 S,P</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 7,401: | Line 7,401: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>9/18 T7-RBS-His- | + | <td>9/18 T7-RBS-His-pSB1C3(1)</td> |
<td>280</td> | <td>280</td> | ||
<td>1.68</td> | <td>1.68</td> | ||
| Line 7,429: | Line 7,429: | ||
<tr> | <tr> | ||
<td>1,2</td> | <td>1,2</td> | ||
| - | <td>9/18 AT- | + | <td>9/18 AT-pSB1C3 X,S</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 7,437: | Line 7,437: | ||
<tr> | <tr> | ||
<td>5,6</td> | <td>5,6</td> | ||
| - | <td>9/18 AT- | + | <td>9/18 AT-pSB1C3 E,P</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 7,709: | Line 7,709: | ||
<img src="https://static.igem.org/mediawiki/2014/2/29/Kyoto-dms-0919_electrophoresis_No2.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"> | <img src="https://static.igem.org/mediawiki/2014/e/e1/Kyoto-dms-0919_electrophoresis_No3.jpg"> | ||
| - | |||
| - | |||
<h4>PCR</h4> | <h4>PCR</h4> | ||
| Line 7,878: | Line 7,876: | ||
</tr> | </tr> | ||
</table> | </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> | <h4>Gel Extraction/Electrophoresis</h4> | ||
| Line 8,082: | Line 8,083: | ||
<tr> | <tr> | ||
<td>5</td> | <td>5</td> | ||
| - | <td>T7-RBS | + | <td>T7-RBS pSB1C3</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 8,290: | Line 8,291: | ||
<tr> | <tr> | ||
<td>33~40</td> | <td>33~40</td> | ||
| - | <td>DiDECARB- | + | <td>DiDECARB-pSB1C3</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>41~48</td> | <td>41~48</td> | ||
| - | <td> | + | <td>pSB1C3 control</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 8,306: | Line 8,307: | ||
<tr> | <tr> | ||
<td>65~72</td> | <td>65~72</td> | ||
| - | <td>REDOX- | + | <td>REDOX-pSB1C3</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 8,314: | Line 8,315: | ||
<tr> | <tr> | ||
<td>81~88</td> | <td>81~88</td> | ||
| - | <td>SAM- | + | <td>SAM-pSB1C3</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 8,393: | Line 8,394: | ||
<tr> | <tr> | ||
<td>8~14</td> | <td>8~14</td> | ||
| - | <td>9/20 SAMmt- | + | <td>9/20 SAMmt-pSB1C3</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 8,401: | Line 8,402: | ||
<tr> | <tr> | ||
<td>22~28</td> | <td>22~28</td> | ||
| - | <td>9/20 DiDECARB- | + | <td>9/20 DiDECARB-pSB1C3</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 8,417: | Line 8,418: | ||
<tr> | <tr> | ||
<td>44</td> | <td>44</td> | ||
| - | <td>9/20 DiDECARB- | + | <td>9/20 DiDECARB-pSB1C3</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 8,466: | Line 8,467: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>HT- | + | <td>HT-pSB1C3 X,P</td> |
<td>4.4</td> | <td>4.4</td> | ||
<td>1.52</td> | <td>1.52</td> | ||
| Line 8,472: | Line 8,473: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>AT- | + | <td>AT-pSB1C3 X,P</td> |
<td>4.0</td> | <td>4.0</td> | ||
<td>1.65</td> | <td>1.65</td> | ||
| Line 8,508: | Line 8,509: | ||
<tr> | <tr> | ||
<td>6</td> | <td>6</td> | ||
| - | <td>AT- | + | <td>AT-pSB1C3 X,P</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>7</td> | <td>7</td> | ||
| - | <td>AT- | + | <td>AT-pSB1C3 X,P</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 8,546: | Line 8,547: | ||
<tr> | <tr> | ||
<td>2~8</td> | <td>2~8</td> | ||
| - | <td>SAMmt- | + | <td>SAMmt-pSB1C3</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 8,576: | Line 8,577: | ||
<tr> | <tr> | ||
<td>2~8</td> | <td>2~8</td> | ||
| - | <td>DiDECARB- | + | <td>DiDECARB-pSB1C3</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 8,633: | Line 8,634: | ||
<tr> | <tr> | ||
<td>5</td> | <td>5</td> | ||
| - | <td>DiDECARB- | + | <td>DiDECARB-pSB1C3-1</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 8,659: | Line 8,660: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>REDOX- | + | <td>REDOX-pSB1C3</td> |
<td>CP</td> | <td>CP</td> | ||
</tr> | </tr> | ||
| Line 8,927: | Line 8,928: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>9/22 DiDECARB | + | <td>9/22 DiDECARB pSB1C3-4</td> |
<td>11.3</td> | <td>11.3</td> | ||
<td>1.54</td> | <td>1.54</td> | ||
| Line 8,933: | Line 8,934: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>9/22 DiDECARB | + | <td>9/22 DiDECARB pSB1C3-2</td> |
<td>10.5</td> | <td>10.5</td> | ||
<td>1.67</td> | <td>1.67</td> | ||
| Line 8,945: | Line 8,946: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>REDOX- | + | <td>REDOX-pSB1C3(2)</td> |
<td>9.8</td> | <td>9.8</td> | ||
<td>1.71</td> | <td>1.71</td> | ||
| Line 8,957: | Line 8,958: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>9/22 DiDECARB | + | <td>9/22 DiDECARB pSB1C3-5</td> |
<td>10.5</td> | <td>10.5</td> | ||
<td>1.69</td> | <td>1.69</td> | ||
| Line 9,134: | Line 9,135: | ||
<tr> | <tr> | ||
<td>15</td> | <td>15</td> | ||
| - | <td>9/15 MP AT- | + | <td>9/15 MP AT-pSB1C3 1</td> |
<td>1.3</td> | <td>1.3</td> | ||
<td>0.5</td> | <td>0.5</td> | ||
| Line 9,145: | Line 9,146: | ||
<tr> | <tr> | ||
<td>16</td> | <td>16</td> | ||
| - | <td>9/15 MP AT- | + | <td>9/15 MP AT-pSB1C3 1</td> |
<td>1.3</td> | <td>1.3</td> | ||
<td>0.5</td> | <td>0.5</td> | ||
| Line 9,156: | Line 9,157: | ||
<tr> | <tr> | ||
<td>17</td> | <td>17</td> | ||
| - | <td>9/15 MP AT- | + | <td>9/15 MP AT-pSB1C3 1</td> |
<td>1.3</td> | <td>1.3</td> | ||
<td>0.5</td> | <td>0.5</td> | ||
| Line 9,167: | Line 9,168: | ||
<tr> | <tr> | ||
<td>18</td> | <td>18</td> | ||
| - | <td>9/15 MP AT- | + | <td>9/15 MP AT-pSB1C3 1</td> |
<td>1.3</td> | <td>1.3</td> | ||
<td>0.5</td> | <td>0.5</td> | ||
| Line 9,178: | Line 9,179: | ||
<tr> | <tr> | ||
<td>19</td> | <td>19</td> | ||
| - | <td>9/22 M REDOX | + | <td>9/22 M REDOX pSB1C3</td> |
<td>0.8</td> | <td>0.8</td> | ||
<td>0.5</td> | <td>0.5</td> | ||
| Line 9,189: | Line 9,190: | ||
<tr> | <tr> | ||
<td>20</td> | <td>20</td> | ||
| - | <td>9/22 M REDOX | + | <td>9/22 M REDOX pSB1C3</td> |
<td>0.8</td> | <td>0.8</td> | ||
<td>0.5</td> | <td>0.5</td> | ||
| Line 9,200: | Line 9,201: | ||
<tr> | <tr> | ||
<td>21</td> | <td>21</td> | ||
| - | <td>9/22 M REDOX | + | <td>9/22 M REDOX pSB1C3</td> |
<td>0.8</td> | <td>0.8</td> | ||
<td>0.5</td> | <td>0.5</td> | ||
| Line 9,211: | Line 9,212: | ||
<tr> | <tr> | ||
<td>22</td> | <td>22</td> | ||
| - | <td>9/18 MP SAM- | + | <td>9/18 MP SAM-pSB1C3</td> |
<td>2.1</td> | <td>2.1</td> | ||
<td>0.5</td> | <td>0.5</td> | ||
| Line 9,222: | Line 9,223: | ||
<tr> | <tr> | ||
<td>23</td> | <td>23</td> | ||
| - | <td>9/18 MP SAM- | + | <td>9/18 MP SAM-pSB1C3</td> |
<td>2.1</td> | <td>2.1</td> | ||
<td>0.5</td> | <td>0.5</td> | ||
| Line 9,233: | Line 9,234: | ||
<tr> | <tr> | ||
<td>24</td> | <td>24</td> | ||
| - | <td>9/18 MP SAM- | + | <td>9/18 MP SAM-pSB1C3</td> |
<td>2.1</td> | <td>2.1</td> | ||
<td>0.5</td> | <td>0.5</td> | ||
| Line 9,244: | Line 9,245: | ||
<tr> | <tr> | ||
<td>25</td> | <td>25</td> | ||
| - | <td>9/22 DiDECARB | + | <td>9/22 DiDECARB pSB1C3-4</td> |
<td>0.7</td> | <td>0.7</td> | ||
<td>0.5</td> | <td>0.5</td> | ||
| Line 9,255: | Line 9,256: | ||
<tr> | <tr> | ||
<td>26</td> | <td>26</td> | ||
| - | <td>9/22 DiDECARB | + | <td>9/22 DiDECARB pSB1C3-4</td> |
<td>0.7</td> | <td>0.7</td> | ||
<td>0.5</td> | <td>0.5</td> | ||
| Line 9,266: | Line 9,267: | ||
<tr> | <tr> | ||
<td>27</td> | <td>27</td> | ||
| - | <td>9/22 DiDECARB | + | <td>9/22 DiDECARB pSB1C3-4</td> |
<td>0.7</td> | <td>0.7</td> | ||
<td>0.5</td> | <td>0.5</td> | ||
| Line 9,277: | Line 9,278: | ||
<tr> | <tr> | ||
<td>28</td> | <td>28</td> | ||
| - | <td>9/22 DiDECARB | + | <td>9/22 DiDECARB pSB1C3-4</td> |
<td>0.7</td> | <td>0.7</td> | ||
<td>0.5</td> | <td>0.5</td> | ||
| Line 10,029: | Line 10,030: | ||
<th>SAMmt Mutation Fw/(µL)</th> | <th>SAMmt Mutation Fw/(µL)</th> | ||
<th>SAMmt Mutation Rv/(µL)</th> | <th>SAMmt Mutation Rv/(µL)</th> | ||
| - | <th>9/18 MP SAM- | + | <th>9/18 MP SAM-pSB1C3/(µL)</th> |
<th>MilliQ/(µL)</th> | <th>MilliQ/(µL)</th> | ||
<th>Total/(µL)</th> | <th>Total/(µL)</th> | ||
| Line 10,250: | Line 10,251: | ||
</tr> | </tr> | ||
</table> | </table> | ||
| - | <p>1. #1 9/15 Miniprep AT- | + | <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>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- | + | <p>3. #1 9/13 Miniprep T7-RBS-pSB1C3 #2 Fw Add His to T7RBS/Rv Add His to T7RBS </p> |
<table> | <table> | ||
<tr> | <tr> | ||
| Line 10,285: | Line 10,286: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>SAMmt- | + | <td>SAMmt-pSB1C3 9/30 column</td> |
<td>136.3</td> | <td>136.3</td> | ||
<td>1.87</td> | <td>1.87</td> | ||
| Line 10,365: | Line 10,366: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>SAMmt- | + | <td>SAMmt-pSB1C3 9/29 Mutation PCR product</td> |
<td>1</td> | <td>1</td> | ||
<td>self-making DH5α</td> | <td>self-making DH5α</td> | ||
| Line 10,373: | Line 10,374: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>AT- | + | <td>AT-pSB1C3 9/30 insertion Mutation PCR product</td> |
<td>1</td> | <td>1</td> | ||
<td>self-making DH5α</td> | <td>self-making DH5α</td> | ||
| Line 10,458: | Line 10,459: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td> | + | <td>pSB1C3 E,P</td> |
<td>5</td> | <td>5</td> | ||
<td>1.78</td> | <td>1.78</td> | ||
| Line 10,475: | Line 10,476: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>9/30 AT- | + | <td>9/30 AT-pSB1C3</td> |
<td>1</td> | <td>1</td> | ||
<td>BL21(DE3)</td> | <td>BL21(DE3)</td> | ||
| Line 10,499: | Line 10,500: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>9/29 SAMmt- | + | <td>9/29 SAMmt-pSB1C3</td> |
<td>1</td> | <td>1</td> | ||
<td>self-making DH5α 2/23</td> | <td>self-making DH5α 2/23</td> | ||
| Line 10,517: | Line 10,518: | ||
</div> | </div> | ||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
<div> | <div> | ||
| - | |||
<a name="1002" class="kyoto-jump"></a> | <a name="1002" class="kyoto-jump"></a> | ||
<h3>10/2</h3> | <h3>10/2</h3> | ||
| Line 10,575: | Line 10,544: | ||
</tr> | </tr> | ||
</table> | </table> | ||
| - | <p>#1 AT- | + | <p>#1 AT-pSB1C3(without (1))</p> |
<p>#2 Fw:Add His to T7RBS/Rv:AT OE-D</p> | <p>#2 Fw:Add His to T7RBS/Rv:AT OE-D</p> | ||
<h4>DNA Purification</h4> | <h4>DNA Purification</h4> | ||
| Line 10,587: | Line 10,556: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>PCR product(3) AT- | + | <td>PCR product(3) AT-pSB1C3 (AT Mota I)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>PCR product(4) AT- | + | <td>PCR product(4) AT-pSB1C3 (AT Mota D)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>PCR product(5) T7-RBS-His- | + | <td>PCR product(5) T7-RBS-His-pSB1C3</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 10,601: | Line 10,570: | ||
<tr> | <tr> | ||
<th>Dpn1/(µL)</th> | <th>Dpn1/(µL)</th> | ||
| - | <th>10/2 PCR AT- | + | <th>10/2 PCR AT-pSB1C3 miniprep product(3)/ |
(µL)</th> | (µL)</th> | ||
<th>10x Buffer(H)/(µL)</th> | <th>10x Buffer(H)/(µL)</th> | ||
| Line 10,624: | Line 10,593: | ||
<tr> | <tr> | ||
<td>4</td> | <td>4</td> | ||
| - | <td>His | + | <td>His pSB1C3-AT 10/2 DNA Purification</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>5</td> | <td>5</td> | ||
| - | <td>AT- | + | <td>AT-pSB1C3 Mutation Deletion 10/2 DNA Purification</ |
td> | td> | ||
</tr> | </tr> | ||
| Line 10,637: | Line 10,606: | ||
<tr> | <tr> | ||
<td>7</td> | <td>7</td> | ||
| - | <td>AT- | + | <td>AT-pSB1C3 Mutation Insert 10/2 DNA Purification</ |
td> | td> | ||
</tr> | </tr> | ||
| Line 10,649: | Line 10,618: | ||
</tr> | </tr> | ||
</table> | </table> | ||
| - | + | <img src="https://static.igem.org/mediawiki/2014/thumb/2/24/IMG-1.jpg/791px-IMG-1.jpg"> | |
</div> | </div> | ||
<div> | <div> | ||
| - | |||
<a name="1003" class="kyoto-jump"></a> | <a name="1003" class="kyoto-jump"></a> | ||
<h3>10/3</h3> | <h3>10/3</h3> | ||
| Line 10,665: | Line 10,633: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>9/14 AT- | + | <td>9/14 AT-pSB1C3(1)</td> |
| - | < | + | <td>119</td> |
| - | < | + | <td>2.00</td> |
| - | < | + | <td>2.19</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>10/1 SAMmt(Mutated)- | + | <td>10/1 SAMmt(Mutated)-pSB1C3(1)</td> |
| - | < | + | <td>275</td> |
| - | < | + | <td>1.86</td> |
| - | < | + | <td>1.75</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>10/1 SAMmt(Mutated)- | + | <td>10/1 SAMmt(Mutated)-pSB1C3(2)</td> |
| - | < | + | <td>227</td> |
| - | < | + | <td>1.93</td> |
| - | < | + | <td>1.90</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>10/1 T7-RBS-His(1)</td> | <td>10/1 T7-RBS-His(1)</td> | ||
| - | < | + | <td>147</td> |
| - | < | + | <td>2.00</td> |
| - | < | + | <td>1.64</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>10/1 T7-RBS-His(2)</td> | <td>10/1 T7-RBS-His(2)</td> | ||
| - | < | + | <td>107</td> |
| - | < | + | <td>1.94</td> |
| - | < | + | <td>1.74</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 10,701: | Line 10,669: | ||
<tr> | <tr> | ||
<th>PrimeSTARPremix/(µL)</th> | <th>PrimeSTARPremix/(µL)</th> | ||
| - | <th>Template 10/3 Miniprep AT- | + | <th>Template 10/3 Miniprep AT-pSB1C3(1)/(µL)</ |
th> | th> | ||
<th>Primer AT Mutation Insert Rv/(µL)</th> | <th>Primer AT Mutation Insert Rv/(µL)</th> | ||
| Line 10,751: | Line 10,719: | ||
</tr> | </tr> | ||
</table> | </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> | <h4>Electrophoresis</h4> | ||
<span class="kyoto-author">Yasuda</span> | <span class="kyoto-author">Yasuda</span> | ||
| Line 10,760: | Line 10,730: | ||
<tr> | <tr> | ||
<td>1</td> | <td>1</td> | ||
| - | <td>AT- | + | <td>AT-pSB1C3 Mutation insert 50.2°C</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>2</td> | <td>2</td> | ||
| - | <td>AT- | + | <td>AT-pSB1C3 Mutation insert 52.8°C</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>3</td> | <td>3</td> | ||
| - | <td>AT- | + | <td>AT-pSB1C3 Mutation insert 54.2°C</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>4</td> | <td>4</td> | ||
| - | <td>AT- | + | <td>AT-pSB1C3 Mutation insert 55.7°C</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>5</td> | <td>5</td> | ||
| - | <td>AT- | + | <td>AT-pSB1C3 Mutation insert 57.1°C</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>6</td> | <td>6</td> | ||
| - | <td>10/2 DNA Purification His | + | <td>10/2 DNA Purification His pSB1C3-AT</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>7</td> | <td>7</td> | ||
| - | <td>AT- | + | <td>AT-pSB1C3 Mutation Deletion 50.2°C</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>8</td> | <td>8</td> | ||
| - | <td>AT- | + | <td>AT-pSB1C3 Mutation Deletion 52.8°C</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>9</td> | <td>9</td> | ||
| - | <td>AT- | + | <td>AT-pSB1C3 Mutation Deletion 54.2°C</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>10</td> | <td>10</td> | ||
| - | <td>AT- | + | <td>AT-pSB1C3 Mutation Deletion 55.7°C</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>11</td> | <td>11</td> | ||
| - | <td>AT- | + | <td>AT-pSB1C3 Mutation Deletion 57.1°C</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 10,807: | Line 10,777: | ||
</tr> | </tr> | ||
</table> | </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> | <h4>OE-PCR 1st</h4> | ||
<span class="kyoto-author">Murata</span> | <span class="kyoto-author">Murata</span> | ||
| Line 10,816: | Line 10,787: | ||
<th>2mM dNTPs/(µL)</th> | <th>2mM dNTPs/(µL)</th> | ||
<th>25mM MgSO4/(µL)</th> | <th>25mM MgSO4/(µL)</th> | ||
| - | <th>10/2 MP J04450( | + | <th>10/2 MP J04450(pSB1C3)/(µL)</th> |
<th>VF2/(µL)</th> | <th>VF2/(µL)</th> | ||
<th>Rv mutation primer T7-RBS-His/(µL)</th> | <th>Rv mutation primer T7-RBS-His/(µL)</th> | ||
| Line 10,841: | Line 10,812: | ||
<th>2mM dNTPs/(µL)</th> | <th>2mM dNTPs/(µL)</th> | ||
<th>25mM MgSO4/(µL)</th> | <th>25mM MgSO4/(µL)</th> | ||
| - | <th>10/2 MP J04450( | + | <th>10/2 MP J04450(pSB1C3)/(µL)</th> |
<th>Fw mutation primer T7-RBS-His/(µL)</th> | <th>Fw mutation primer T7-RBS-His/(µL)</th> | ||
<th>Present from Santa/(µL)</th> | <th>Present from Santa/(µL)</th> | ||
| Line 10,937: | Line 10,908: | ||
</table> | </table> | ||
<p>Denature-Annealing-Extension 35cycle</p> | <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> | <h4>Restriction Enzyme Digestion</h4> | ||
<span class="kyoto-author">Yasuda</span> | <span class="kyoto-author">Yasuda</span> | ||
| Line 10,951: | Line 10,924: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>AT- | + | <td>AT-pSB1C3(1) 10/3 Miniprep</td> |
<td>10</td> | <td>10</td> | ||
<td>1</td> | <td>1</td> | ||
<td>1</td> | <td>1</td> | ||
| - | |||
<td>4</td> | <td>4</td> | ||
<td>4</td> | <td>4</td> | ||
| Line 10,967: | Line 10,939: | ||
<td>1</td> | <td>1</td> | ||
<td>1</td> | <td>1</td> | ||
| - | |||
<td>4</td> | <td>4</td> | ||
<td>4</td> | <td>4</td> | ||
| Line 10,987: | Line 10,958: | ||
<th>Total/(µL)</tr> | <th>Total/(µL)</tr> | ||
<tr> | <tr> | ||
| - | <td> | + | <td>pSB1C3 10/3 XA</td> |
<td>2</td> | <td>2</td> | ||
<td>T7 RBS His 10/3 XA</td> | <td>T7 RBS His 10/3 XA</td> | ||
| Line 10,999: | Line 10,970: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td> | + | <td>pSB1C3 10/3 XA</td> |
<td>2</td> | <td>2</td> | ||
<td>T7 RBS His 10/3 XA</td> | <td>T7 RBS His 10/3 XA</td> | ||
| Line 11,020: | Line 10,991: | ||
<th>Total/(µL)</tr> | <th>Total/(µL)</tr> | ||
<tr> | <tr> | ||
| - | <td> | + | <td>pSB1C3 10/3 XA</td> |
<td>2</td> | <td>2</td> | ||
<td>T7 RBS His 10/3 XA</td> | <td>T7 RBS His 10/3 XA</td> | ||
| Line 11,030: | Line 11,001: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td> | + | <td>pSB1C3 10/3 XA</td> |
<td>1</td> | <td>1</td> | ||
<td>T7 RBS His 10/3 XA</td> | <td>T7 RBS His 10/3 XA</td> | ||
| Line 11,054: | Line 11,025: | ||
</tr> | </tr> | ||
</table> | </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> | ||
<div> | <div> | ||
| - | + | ||
<a name="1004" class="kyoto-jump"></a> | <a name="1004" class="kyoto-jump"></a> | ||
<h3>10/4</h3> | <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> | ||
<div> | <div> | ||
| - | + | ||
<a name="1005" class="kyoto-jump"></a> | <a name="1005" class="kyoto-jump"></a> | ||
<h3>10/5</h3> | <h3>10/5</h3> | ||
| Line 11,082: | Line 11,214: | ||
<td>2</td> | <td>2</td> | ||
<td>10</td> | <td>10</td> | ||
| - | |||
<td>10</td> | <td>10</td> | ||
<td>6</td> | <td>6</td> | ||
| Line 11,161: | Line 11,292: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>10/5 | + | <td>10/5 pSB1C3 XA (1) 9/18 Mutated Bn-pSB1C310-3</td> |
<td>5</td> | <td>5</td> | ||
<td>1</td> | <td>1</td> | ||
| Line 11,172: | Line 11,303: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>10/5 | + | <td>10/5 pSB1C3 XA (2) 9/14 Mutated |
| - | dddD(mutated)- | + | dddD(mutated)-pSB1C3(2)</td> |
<td>5</td> | <td>5</td> | ||
<td>1</td> | <td>1</td> | ||
| Line 11,201: | Line 11,332: | ||
<tr> | <tr> | ||
<td>(1)-4,5</td> | <td>(1)-4,5</td> | ||
| - | <td> | + | <td>pSB1C3 10/5 XA(2)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 11,217: | Line 11,348: | ||
<tr> | <tr> | ||
<td>(2)-4,5</td> | <td>(2)-4,5</td> | ||
| - | <td> | + | <td>pSB1C3 10/5 XA(1)</td> |
</tr> | </tr> | ||
</table> | </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> | <div> | ||
| - | |||
<a name="1006" class="kyoto-jump"></a> | <a name="1006" class="kyoto-jump"></a> | ||
<h3>10/6</h3> | <h3>10/6</h3> | ||
| Line 11,441: | Line 11,687: | ||
</div> | </div> | ||
<div> | <div> | ||
| - | + | ||
<a name="1007" class="kyoto-jump"></a> | <a name="1007" class="kyoto-jump"></a> | ||
<h3>10/7</h3> | <h3>10/7</h3> | ||
| Line 11,688: | Line 11,934: | ||
</tr> | </tr> | ||
</table> | </table> | ||
| - | + | ||
</div> | </div> | ||
<div> | <div> | ||
| - | + | ||
<a name="1008" class="kyoto-jump"></a> | <a name="1008" class="kyoto-jump"></a> | ||
<h3>10/8</h3> | <h3>10/8</h3> | ||
| Line 11,714: | Line 11,960: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>9/30 AT- | + | <td>9/30 AT-pSB1C3 insertion mutated</td> |
<td>CP</td> | <td>CP</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>9/30 SAMmt- | + | <td>9/30 SAMmt-pSB1C3 mutated</td> |
<td>CP</td> | <td>CP</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
| - | + | ||
</div> | </div> | ||
| - | + | ||
<div> | <div> | ||
| - | + | ||
<a name="1010" class="kyoto-jump"></a> | <a name="1010" class="kyoto-jump"></a> | ||
<h3>10/10</h3> | <h3>10/10</h3> | ||
| Line 11,744: | Line 11,990: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>AT(Mutation Insert)- | + | <td>AT(Mutation Insert)-pSB1C3(2)</td> |
<td>20</td> | <td>20</td> | ||
<td>-</td> | <td>-</td> | ||
| Line 11,795: | Line 12,041: | ||
<tr> | <tr> | ||
<td>Liquid Culture product | <td>Liquid Culture product | ||
| - | SAM(Mutated)- | + | SAM(Mutated)-pSB1C3(1)~(4)</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 11,805: | Line 12,051: | ||
<th>AT OE-A/(µL)</th> | <th>AT OE-A/(µL)</th> | ||
<th>AT cloning for His/(µL)</th> | <th>AT cloning for His/(µL)</th> | ||
| - | <th>10/7 M AT(Mutation Insert)- | + | <th>10/7 M AT(Mutation Insert)-pSB1C3(2)/(µL)</ |
th> | th> | ||
<th>MilliQ/(µL)</th> | <th>MilliQ/(µL)</th> | ||
| Line 11,852: | Line 12,098: | ||
<tr> | <tr> | ||
<td>1</td> | <td>1</td> | ||
| - | <td>AT | + | <td>AT pSB1C3 (Mutation Insert)(2)10/3</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 11,879: | Line 12,125: | ||
<tr> | <tr> | ||
<td>10/10 Miniprep product | <td>10/10 Miniprep product | ||
| - | SAMmt(Mutated)- | + | SAMmt(Mutated)-pSB1C3(1)</td> |
<td>10</td> | <td>10</td> | ||
<td>-</td> | <td>-</td> | ||
| Line 11,893: | Line 12,139: | ||
<tr> | <tr> | ||
<td>10/10 Miniprep product | <td>10/10 Miniprep product | ||
| - | SAMmt(Mutated)- | + | SAMmt(Mutated)-pSB1C3(2)</td> |
<td>10</td> | <td>10</td> | ||
<td>-</td> | <td>-</td> | ||
| Line 11,907: | Line 12,153: | ||
<tr> | <tr> | ||
<td>10/10 Miniprep product | <td>10/10 Miniprep product | ||
| - | SAMmt(Mutated)- | + | SAMmt(Mutated)-pSB1C3(3)</td> |
<td>10</td> | <td>10</td> | ||
<td>-</td> | <td>-</td> | ||
| Line 11,921: | Line 12,167: | ||
<tr> | <tr> | ||
<td>10/10 Miniprep product | <td>10/10 Miniprep product | ||
| - | SAMmt(Mutated)- | + | SAMmt(Mutated)-pSB1C3(4)</td> |
<td>10</td> | <td>10</td> | ||
<td>-</td> | <td>-</td> | ||
| Line 11,945: | Line 12,191: | ||
<th>Total/(µL)</tr> | <th>Total/(µL)</tr> | ||
<tr> | <tr> | ||
| - | <td>T7 RBS- | + | <td>T7 RBS-pSB1C3 S,P</td> |
<td>2</td> | <td>2</td> | ||
<td>10/10 gel extraction product(true)XP</td> | <td>10/10 gel extraction product(true)XP</td> | ||
| Line 11,957: | Line 12,203: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>T7 RBS- | + | <td>T7 RBS-pSB1C3 S,P</td> |
<td>1</td> | <td>1</td> | ||
<td>10/10 gel extraction product(true)XP</td> | <td>10/10 gel extraction product(true)XP</td> | ||
| Line 11,969: | Line 12,215: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>T7 RBS- | + | <td>T7 RBS-pSB1C3 S,P</td> |
<td>2</td> | <td>2</td> | ||
<td>10/10 gel extraction product(true)XP</td> | <td>10/10 gel extraction product(true)XP</td> | ||
| Line 11,981: | Line 12,227: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>T7 RBS- | + | <td>T7 RBS-pSB1C3 S,P</td> |
<td>2</td> | <td>2</td> | ||
<td>10/10 gel extraction product(true)XP</td> | <td>10/10 gel extraction product(true)XP</td> | ||
| Line 11,993: | Line 12,239: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>T7 RBS- | + | <td>T7 RBS-pSB1C3 S,P</td> |
<td>1</td> | <td>1</td> | ||
<td>10/10 gel extraction product(true)XP</td> | <td>10/10 gel extraction product(true)XP</td> | ||
| Line 12,005: | Line 12,251: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>T7 RBS- | + | <td>T7 RBS-pSB1C3 S,P</td> |
<td>2</td> | <td>2</td> | ||
<td>10/10 gel extraction product(true)XP</td> | <td>10/10 gel extraction product(true)XP</td> | ||
| Line 12,031: | Line 12,277: | ||
</tr> | </tr> | ||
</table> | </table> | ||
| - | + | ||
</div> | </div> | ||
<div> | <div> | ||
| - | + | ||
<a name="1011" class="kyoto-jump"></a> | <a name="1011" class="kyoto-jump"></a> | ||
<h3>10/11</h3> | <h3>10/11</h3> | ||
| Line 12,059: | Line 12,305: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>AT- | + | <td>AT-pSB1C3(2)</td> |
<td>258.0</td> | <td>258.0</td> | ||
<td>1.96</td> | <td>1.96</td> | ||
| Line 12,076: | Line 12,322: | ||
<th colspan=2>Buffer/(µL)</th> | <th colspan=2>Buffer/(µL)</th> | ||
<th>BSA/(µL)</th> | <th>BSA/(µL)</th> | ||
| - | < | + | <th>CIAP/(µL)</th> |
<th>MilliQ/(µL)</th> | <th>MilliQ/(µL)</th> | ||
<th>Total/(µL)</th> | <th>Total/(µL)</th> | ||
| Line 12,107: | Line 12,353: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>10/11 miniprep product AT- | + | <td>10/11 miniprep product AT-pSB1C3(2)</td> |
<td>10</td> | <td>10</td> | ||
<td>-</td> | <td>-</td> | ||
| Line 12,121: | Line 12,367: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>10/10 miniprep product SAM(mutated)- | + | <td>10/10 miniprep product SAM(mutated)-pSB1C3-(1)</ |
td> | td> | ||
<td>10</td> | <td>10</td> | ||
| Line 12,136: | Line 12,382: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>10/10 miniprep product SAM(mutated)- | + | <td>10/10 miniprep product SAM(mutated)-pSB1C3-(2)</ |
td> | td> | ||
<td>10</td> | <td>10</td> | ||
| Line 12,151: | Line 12,397: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>10/10 miniprep product SAM(mutated)- | + | <td>10/10 miniprep product SAM(mutated)-pSB1C3-(3)</ |
td> | td> | ||
<td>10</td> | <td>10</td> | ||
| Line 12,166: | Line 12,412: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td>10/10 miniprep product SAM(mutated)- | + | <td>10/10 miniprep product SAM(mutated)-pSB1C3-(4)</ |
td> | td> | ||
<td>10</td> | <td>10</td> | ||
| Line 12,231: | Line 12,477: | ||
<th>Sample</th> | <th>Sample</th> | ||
<th>CompetentCells</th> | <th>CompetentCells</th> | ||
| - | |||
<th>Medium</th> | <th>Medium</th> | ||
</tr> | </tr> | ||
| Line 12,241: | Line 12,486: | ||
</table> | </table> | ||
</div> | </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> | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
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 |
| 6 | 1kbp GeneRuler |
| 7 | AT-pSB1C3 Mutation Insert 10/2 DNA Purification |
| 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) | 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) | 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) | 10 | - | - | 1 | - | B | 4 | 0.4 | 0 | 24.6 | 40 |
| 10/10 miniprep product SAM(mutated)-pSB1C3-(2) | 10 | - | - | 1 | - | B | 4 | 0.4 | 0 | 24.6 | 40 |
| 10/10 miniprep product SAM(mutated)-pSB1C3-(3) | 10 | - | - | 1 | - | B | 4 | 0.4 | 0 | 24.6 | 40 |
| 10/10 miniprep product SAM(mutated)-pSB1C3-(4) | 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 |
