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Team:KIT-Kyoto/Notebook/Labnote
From 2014.igem.org
(Difference between revisions)
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<li class="category"> | <li class="category"> | ||
<a href="javascript:void(0)"> | <a href="javascript:void(0)"> | ||
| - | <font color="#143"> | + | <font color="#143">Click Here To Enlarge</font> LabNote |
</a> | </a> | ||
</li> | </li> | ||
| Line 220: | Line 220: | ||
<td>pGEX6P-2</td> | <td>pGEX6P-2</td> | ||
<td>CuMTSE1</td> | <td>CuMTSE1</td> | ||
| - | <td> | + | <td>Non Treated</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 241: | Line 241: | ||
<td>pGEX6P-2</td> | <td>pGEX6P-2</td> | ||
<td>CuMTSE2</td> | <td>CuMTSE2</td> | ||
| - | <td> | + | <td>Non Treated</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 262: | Line 262: | ||
<td>pGEX6P-2</td> | <td>pGEX6P-2</td> | ||
<td>CuMTS3</td> | <td>CuMTS3</td> | ||
| - | <td> | + | <td>Non Treated</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 283: | Line 283: | ||
<td>pGEX6P-2</td> | <td>pGEX6P-2</td> | ||
<td>CuMTS62</td> | <td>CuMTS62</td> | ||
| - | <td> | + | <td>Non Treated</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 371: | Line 371: | ||
<th colspan="2">Sample</th> | <th colspan="2">Sample</th> | ||
<th rowspan="2">Section</th> | <th rowspan="2">Section</th> | ||
| - | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 756: | Line 755: | ||
<!--AGE--> | <!--AGE--> | ||
<br> | <br> | ||
| - | + | Apply λDNA-HindⅢ to compare and contrast AGE of July 14 and June 11. | |
<br> | <br> | ||
<img src="/wiki/images/b/b6/Kit_June14_c.jpg"> | <img src="/wiki/images/b/b6/Kit_June14_c.jpg"> | ||
| Line 972: | Line 971: | ||
<!--Main Culture終わり--> | <!--Main Culture終わり--> | ||
<br> | <br> | ||
| - | <!--Protein Extraction (<em>E.coli</em>)--> | + | <!--Protein Extraction (<em>E. coli</em>)--> |
| - | <strong>Protein Extraction (<em>E.coli</em>)</strong> | + | <strong>Protein Extraction (<em>E. coli</em>)</strong> |
<div class="m_table"> | <div class="m_table"> | ||
<table class="materials"> | <table class="materials"> | ||
| Line 1,021: | Line 1,020: | ||
</table> | </table> | ||
</div> | </div> | ||
| - | <!--Protein Extraction (<em>E.coli</em>)終わり--> | + | <!--Protein Extraction (<em>E. coli</em>)終わり--> |
<br> | <br> | ||
<!--Preparations for SDS-PAGE--> | <!--Preparations for SDS-PAGE--> | ||
| Line 1,300: | Line 1,299: | ||
<!-- 実験者名終わり --> | <!-- 実験者名終わり --> | ||
<br> | <br> | ||
| - | + | Cultivate following samples in Big Scale at 30℃ overnight in shaken culture. | |
<br> | <br> | ||
<div class="m_table"> | <div class="m_table"> | ||
| Line 1,332: | Line 1,331: | ||
<br> | <br> | ||
| - | <!--DNA | + | <!--DNA Refinement 1--> |
| - | <strong>DNA | + | <strong>DNA Refinement 1</strong> |
<div class="m_table"> | <div class="m_table"> | ||
<table class="materials"> | <table class="materials"> | ||
| Line 1,352: | Line 1,351: | ||
</table> | </table> | ||
</div> | </div> | ||
| - | <!--DNA | + | <!--DNA Refinement 1終わり--> |
<br> | <br> | ||
| Line 1,397: | Line 1,396: | ||
<br> | <br> | ||
| - | <!--DNA | + | <!--DNA Refinement 2--> |
| - | <strong>DNA | + | <strong>DNA Refinement 2</strong> |
<div class="m_table"> | <div class="m_table"> | ||
<table class="materials"> | <table class="materials"> | ||
| Line 1,430: | Line 1,429: | ||
</table> | </table> | ||
</div> | </div> | ||
| - | <!--DNA | + | <!--DNA Refinement 2--> |
<br> | <br> | ||
| Line 1,985: | Line 1,984: | ||
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
| - | <td> | + | <td>Non Treated</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 2,017: | Line 2,016: | ||
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
| - | <td> | + | <td>Non Treated</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 2,041: | Line 2,040: | ||
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
| - | <td> | + | <td>Non Treated</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 2,072: | Line 2,071: | ||
<td>p427-TEF</td> | <td>p427-TEF</td> | ||
<td>CuMTSE1</td> | <td>CuMTSE1</td> | ||
| - | <td>pGEX6P-2F2, pGEX6P-2R<td> | + | <td>pGEX6P-2F2, pGEX6P-2R</td> |
</tr> | </tr> | ||
| - | |||
</table> | </table> | ||
</div> | </div> | ||
| Line 2,153: | Line 2,151: | ||
KOBAYASHI<br> | KOBAYASHI<br> | ||
<!-- 実験者名終わり --> | <!-- 実験者名終わり --> | ||
| - | <!--Protein Extraction (<em> | + | <!--Protein Extraction (<em>S. cerevisiae</em>)--> |
| - | <strong>Protein Extraction (<em> | + | <strong>Protein Extraction (<em>S. cerevisiae</em>)</strong> |
<div class="m_table"> | <div class="m_table"> | ||
<table class="materials"> | <table class="materials"> | ||
| Line 2,180: | Line 2,178: | ||
</table> | </table> | ||
</div> | </div> | ||
| - | <!--Protein Extraction (<em> | + | <!--Protein Extraction (<em>S. cerevisiae</em>)--> |
<br> | <br> | ||
| Line 2,284: | Line 2,282: | ||
<td>p427-TEF</td> | <td>p427-TEF</td> | ||
<td>CuMTSE1</td> | <td>CuMTSE1</td> | ||
| - | <td>pGEX6P-2F2,pGEX6P-2R</td> | + | <td>pGEX6P-2F2, pGEX6P-2R</td> |
<td><em>Sac</em>1, <em>Kpn</em>1</td> | <td><em>Sac</em>1, <em>Kpn</em>1</td> | ||
<td>Buffer L</td> | <td>Buffer L</td> | ||
| Line 2,315: | Line 2,313: | ||
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
| - | <td> | + | <td>Non Treated</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 2,323: | Line 2,321: | ||
<td>CuMTSE1</td> | <td>CuMTSE1</td> | ||
<td>pGEX6P-2F1, pGEX6P-2R</td> | <td>pGEX6P-2F1, pGEX6P-2R</td> | ||
| - | <td> | + | <td>Non Treated</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 2,331: | Line 2,329: | ||
<td>CuMTSE1</td> | <td>CuMTSE1</td> | ||
<td>pGEX6P-2F2, pGEX6P-2R</td> | <td>pGEX6P-2F2, pGEX6P-2R</td> | ||
| - | <td> | + | <td>Non Treated</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 2,488: | Line 2,486: | ||
<td>p427-TEF</td> | <td>p427-TEF</td> | ||
<td>CuMTSE1</td> | <td>CuMTSE1</td> | ||
| - | <td>pGEX6P-2F2,pGEX6P-2R</td> | + | <td>pGEX6P-2F2, pGEX6P-2R</td> |
<td><em>Sac</em>1</td> | <td><em>Sac</em>1</td> | ||
<td>Buffer L</td> | <td>Buffer L</td> | ||
| Line 2,731: | Line 2,729: | ||
<br> | <br> | ||
| - | <!--Protein Extraction (<em> | + | <!--Protein Extraction (<em>S. cerevisiae</em>)--> |
| - | <strong>Protein Extraction (<em> | + | <strong>Protein Extraction (<em>S. cerevisiae</em>)</strong> |
<br> | <br> | ||
<div class="m_table"> | <div class="m_table"> | ||
| Line 2,765: | Line 2,763: | ||
</table> | </table> | ||
</div> | </div> | ||
| - | <!--Protein Extraction (<em> | + | <!--Protein Extraction (<em>S. cerevisiae</em>)--> |
<br> | <br> | ||
| Line 3,029: | Line 3,027: | ||
<td>六</td> | <td>六</td> | ||
<td>p427-TEF</td> | <td>p427-TEF</td> | ||
| - | <td> | + | <td>CuMTSE1</td> |
<td>pGEX6P-2F2, pGEX6P-2R</td> | <td>pGEX6P-2F2, pGEX6P-2R</td> | ||
</tr> | </tr> | ||
| Line 3,049: | Line 3,047: | ||
SOGA<br> | SOGA<br> | ||
<!-- 実験者名終わり --> | <!-- 実験者名終わり --> | ||
| - | <!--DNA | + | <!--DNA Refinement 1--> |
| - | <strong>DNA | + | <strong>DNA Refinement 1</strong> |
<br> | <br> | ||
<div class="m_table"> | <div class="m_table"> | ||
| Line 3,078: | Line 3,076: | ||
</table> | </table> | ||
</div> | </div> | ||
| - | <!--DNA | + | <!--DNA Refinement 1--> |
<br> | <br> | ||
| Line 3,135: | Line 3,133: | ||
<br> | <br> | ||
| - | <!--DNA | + | <!--DNA Refinement 2--> |
| - | <strong>DNA | + | <strong>DNA Refinement 2</strong> |
<div class="m_table"> | <div class="m_table"> | ||
<table class="materials"> | <table class="materials"> | ||
| Line 3,159: | Line 3,157: | ||
</table> | </table> | ||
</div> | </div> | ||
| - | <!--DNA | + | <!--DNA Refinement 2--> |
<br> | <br> | ||
| Line 3,193: | Line 3,191: | ||
DNA ligase: DNA Ligation Kit ‹Mighty Mix› (TAKARA BIO INC.) | DNA ligase: DNA Ligation Kit ‹Mighty Mix› (TAKARA BIO INC.) | ||
<br> | <br> | ||
| - | <!--DNA | + | <!--DNA Refinement 2--> |
<br> | <br> | ||
| - | <!--Transformation (<em>E.coli</em>)--> | + | <!--Transformation (<em>E. coli</em>)--> |
| - | <strong>Transformation (<em>E.coli</em>)</strong> | + | <strong>Transformation (<em>E. coli</em>)</strong> |
<br> | <br> | ||
<div class="m_table"> | <div class="m_table"> | ||
| Line 3,242: | Line 3,240: | ||
</table> | </table> | ||
</div> | </div> | ||
| - | <!--Transformation (<em>E.coli</em>)--> | + | <!--Transformation (<em>E. coli</em>)--> |
</p> | </p> | ||
<!--本文終わり--> | <!--本文終わり--> | ||
| Line 3,302: | Line 3,300: | ||
<br> | <br> | ||
| - | <!--Transformation (<em>E.coli</em>)--> | + | <!--Transformation (<em>E. coli</em>)--> |
| - | <strong>Transformation (<em>E.coli</em>)</strong> | + | <strong>Transformation (<em>E. coli</em>)</strong> |
<br> | <br> | ||
<div class="m_table"> | <div class="m_table"> | ||
| Line 3,326: | Line 3,324: | ||
</table> | </table> | ||
</div> | </div> | ||
| - | <!--Transformation (<em>E.coli</em>)--> | + | <!--Transformation (<em>E. coli</em>)--> |
<br> | <br> | ||
| Line 3,752: | Line 3,750: | ||
<td>CuMTS3</td> | <td>CuMTS3</td> | ||
<td>pGEX6P-2F1, pGEX6P-2R</td> | <td>pGEX6P-2F1, pGEX6P-2R</td> | ||
| - | <td> | + | <td>Non Treated</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 3,766: | Line 3,764: | ||
<td>CuMTS3</td> | <td>CuMTS3</td> | ||
<td>pGEX6P-2F1, pGEX6P-2R</td> | <td>pGEX6P-2F1, pGEX6P-2R</td> | ||
| - | <td> | + | <td>Non Treated</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 3,780: | Line 3,778: | ||
<td>CuMTS3</td> | <td>CuMTS3</td> | ||
<td>pGEX6P-2F1, pGEX6P-2R</td> | <td>pGEX6P-2F1, pGEX6P-2R</td> | ||
| - | <td> | + | <td>Non Treated</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 3,794: | Line 3,792: | ||
<td>CuMTS62</td> | <td>CuMTS62</td> | ||
<td>pGEX6P-2F1, pGEX6P-2R</td> | <td>pGEX6P-2F1, pGEX6P-2R</td> | ||
| - | <td> | + | <td>Non Treated</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 3,808: | Line 3,806: | ||
<td>CuMTS62</td> | <td>CuMTS62</td> | ||
<td>pGEX6P-2F1, pGEX6P-2R</td> | <td>pGEX6P-2F1, pGEX6P-2R</td> | ||
| - | <td> | + | <td>Non Treated</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 3,822: | Line 3,820: | ||
<td>CuMTS62</td> | <td>CuMTS62</td> | ||
<td>pGEX6P-2F1, pGEX6P-2R</td> | <td>pGEX6P-2F1, pGEX6P-2R</td> | ||
| - | <td> | + | <td>Non Treated</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 3,848: | Line 3,846: | ||
<h3>August 19, 2014</h3> | <h3>August 19, 2014</h3> | ||
<p class="sentence"> | <p class="sentence"> | ||
| - | + | KOBAYASHI | |
| + | <br> | ||
| + | Conduct mutagenesis to remove an illegal restriction site (<em>Eco</em>R1) | ||
<br> | <br> | ||
<strong>InversePCR</strong> | <strong>InversePCR</strong> | ||
| Line 3,860: | Line 3,860: | ||
<th>Insert</th> | <th>Insert</th> | ||
<th>Primer</th> | <th>Primer</th> | ||
| - | <th>DNA | + | <th>DNA polymerase</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>pGEX6P-2</td> | <td>pGEX6P-2</td> | ||
<td>CuMTS3</td> | <td>CuMTS3</td> | ||
| - | <td> | + | <td>Terpinene3-mutagenesis-FP, Terpinene3-mutagenesis-RP</td> |
<td>KOD-Plus</td> | <td>KOD-Plus</td> | ||
</tr> | </tr> | ||
| Line 3,871: | Line 3,871: | ||
</div> | </div> | ||
(TOYOBO, KOD-Plus-Mutagenesis Kit) | (TOYOBO, KOD-Plus-Mutagenesis Kit) | ||
| - | + | Start from KOD -Plus- Mutagenesis Kit protocol No.2. | |
<br> | <br> | ||
<br> | <br> | ||
| Line 3,879: | Line 3,879: | ||
<h3>August 20, 2014</h3> | <h3>August 20, 2014</h3> | ||
<p class="sentence"> | <p class="sentence"> | ||
| - | + | KOBAYASHI | |
| + | <br> | ||
| + | Restart mutagenesis from KOD -Plus- Mutagenesis Kit protocol No.3 to obtain plasmid (muta-CuMTS3 in pGEX-6P-2) | ||
<br> | <br> | ||
<strong>Transformation</strong> | <strong>Transformation</strong> | ||
| Line 3,896: | Line 3,898: | ||
<td rowspan="2">DH5α</td> | <td rowspan="2">DH5α</td> | ||
<td rowspan="2">pGEX6P-2</td> | <td rowspan="2">pGEX6P-2</td> | ||
| - | <td rowspan="2"> | + | <td rowspan="2">muta-CuMTS3</td> |
| - | <td>Terpinene3-mutagenesis-FP and | + | <td>Terpinene3-mutagenesis-FP and Terpinene3-mutagenesis-RP</td> |
| - | + | ||
| - | + | ||
| - | + | ||
</tr> | </tr> | ||
</table> | </table> | ||
| Line 3,910: | Line 3,909: | ||
<h3>August 21, 2014</h3> | <h3>August 21, 2014</h3> | ||
<p class="sentence"> | <p class="sentence"> | ||
| + | KOBAYASHI | ||
| + | <br> | ||
<strong>Colony Isolation</strong><br> | <strong>Colony Isolation</strong><br> | ||
| - | + | Isolate each colony from two plates which were inoculated overnight | |
<div class="m_table"> | <div class="m_table"> | ||
<table class="materials"> | <table class="materials"> | ||
<tr> | <tr> | ||
<th colspan="4">Sample</th> | <th colspan="4">Sample</th> | ||
| - | <th></th> | + | <th rowspan="2">Section</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 3,923: | Line 3,924: | ||
<th>Insert</th> | <th>Insert</th> | ||
<th>Primer</th> | <th>Primer</th> | ||
| - | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>DH5α</td> | <td>DH5α</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td>Terpinene3-mutagenesis-FP,<br>Terpinene3-mutagenesis-RP</td> | <td>Terpinene3-mutagenesis-FP,<br>Terpinene3-mutagenesis-RP</td> | ||
<td>あ-み</td> | <td>あ-み</td> | ||
| Line 3,934: | Line 3,934: | ||
</table> | </table> | ||
</div> | </div> | ||
| - | + | Select 5 isolated colonies whose code names are あ,か,さ,た and な and miniprep them. | |
<br> | <br> | ||
<br> | <br> | ||
| Line 3,941: | Line 3,941: | ||
<h3>August 22, 2014</h3> | <h3>August 22, 2014</h3> | ||
<p class="sentence"> | <p class="sentence"> | ||
| + | KOBAYASHI | ||
| + | <br> | ||
<strong>Restriction Digest</strong> | <strong>Restriction Digest</strong> | ||
<div class="m_table"> | <div class="m_table"> | ||
<table class="materials"> | <table class="materials"> | ||
<tr> | <tr> | ||
| - | <th>Sample</th> | + | <th colspan="3">Sample</th> |
| - | <th></th> | + | <th rowspan="2">Enzyme</th> |
| - | <th></th> | + | <th rowspan="2">Buffer</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 3,953: | Line 3,955: | ||
<th>Vector</th> | <th>Vector</th> | ||
<th>Insert</th> | <th>Insert</th> | ||
| - | |||
| - | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>DH5α あ</td> | <td>DH5α あ</td> | ||
<td>pGEX6P-2</td> | <td>pGEX6P-2</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Sal</em>1</td> | <td><em>Sal</em>1</td> | ||
| - | <td>Fast | + | <td>Fast Digest buffer</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>DH5α か</td> | <td>DH5α か</td> | ||
<td>pGEX6P-2</td> | <td>pGEX6P-2</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Sal</em>1</td> | <td><em>Sal</em>1</td> | ||
| - | <td>Fast | + | <td>Fast Digest buffer</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>DH5α さ</td> | <td>DH5α さ</td> | ||
<td>pGEX6P-2</td> | <td>pGEX6P-2</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Sal</em>1</td> | <td><em>Sal</em>1</td> | ||
| - | <td>Fast | + | <td>Fast Digest buffer</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>DH5α た</td> | <td>DH5α た</td> | ||
<td>pGEX6P-2</td> | <td>pGEX6P-2</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Sal</em>1</td> | <td><em>Sal</em>1</td> | ||
| - | <td>Fast | + | <td>Fast Digest buffer</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 3,992: | Line 3,992: | ||
<table class="materials"> | <table class="materials"> | ||
<tr> | <tr> | ||
| - | <th></th> | + | <th rowspan="2">Lane</th> |
<th colspan="4">Sample</th> | <th colspan="4">Sample</th> | ||
| - | <th></th> | + | <th rowspan="2">Enzyme</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | |||
<th>Marker</th> | <th>Marker</th> | ||
<th>Host</th> | <th>Host</th> | ||
<th>Vector</th> | <th>Vector</th> | ||
<th>Insert</th> | <th>Insert</th> | ||
| - | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,017: | Line 4,015: | ||
<td>DH5α あ</td> | <td>DH5α あ</td> | ||
<td>pGEX6P-2</td> | <td>pGEX6P-2</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Sal</em>1</td> | <td><em>Sal</em>1</td> | ||
</tr> | </tr> | ||
| Line 4,025: | Line 4,023: | ||
<td>DH5α か</td> | <td>DH5α か</td> | ||
<td>pGEX6P-2</td> | <td>pGEX6P-2</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Sal</em>1</td> | <td><em>Sal</em>1</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td> | + | <td>4</td> |
<td></td> | <td></td> | ||
<td>DH5α さ</td> | <td>DH5α さ</td> | ||
<td>pGEX6P-2</td> | <td>pGEX6P-2</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Sal</em>1</td> | <td><em>Sal</em>1</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td> | + | <td>5</td> |
<td></td> | <td></td> | ||
<td>DH5α た</td> | <td>DH5α た</td> | ||
<td>pGEX6P-2</td> | <td>pGEX6P-2</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Sal</em>1</td> | <td><em>Sal</em>1</td> | ||
</tr> | </tr> | ||
| Line 4,052: | Line 4,050: | ||
<tr> | <tr> | ||
<th colspan="3">Sample</th> | <th colspan="3">Sample</th> | ||
| - | <th></th> | + | <th rowspan="2">Enzyme</th> |
| - | <th></th> | + | <th rowspan="2">Buffer</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,059: | Line 4,057: | ||
<th>Vector</th> | <th>Vector</th> | ||
<th>Insert</th> | <th>Insert</th> | ||
| - | |||
| - | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,067: | Line 4,063: | ||
<td>CuMTS3</td> | <td>CuMTS3</td> | ||
<td><em>Eco</em>R1</td> | <td><em>Eco</em>R1</td> | ||
| - | <td>Fast | + | <td>Fast Digest buffer</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>DH5α か</td> | <td>DH5α か</td> | ||
<td>pGEX6P-2</td> | <td>pGEX6P-2</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Eco</em>R1</td> | <td><em>Eco</em>R1</td> | ||
| - | <td>Fast | + | <td>Fast Digest buffer</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 4,083: | Line 4,079: | ||
<table class="materials"> | <table class="materials"> | ||
<tr> | <tr> | ||
| - | <th | + | <th rowspan="2">Lane</th> |
<th colspan="4">Sample</th> | <th colspan="4">Sample</th> | ||
| - | <th></th> | + | <th rowspan="2">Enzyme</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,092: | Line 4,088: | ||
<th>Vector</th> | <th>Vector</th> | ||
<th>Insert</th> | <th>Insert</th> | ||
| - | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,104: | Line 4,099: | ||
<tr> | <tr> | ||
<td>2</td> | <td>2</td> | ||
| + | <td></td> | ||
<td>DH5α か</td> | <td>DH5α か</td> | ||
<td>pGEX6P-2</td> | <td>pGEX6P-2</td> | ||
| Line 4,111: | Line 4,107: | ||
<tr> | <tr> | ||
<td>3</td> | <td>3</td> | ||
| + | <td></td> | ||
<td>DH5α か</td> | <td>DH5α か</td> | ||
<td>pGEX6P-2</td> | <td>pGEX6P-2</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Eco</em>R1</td> | <td><em>Eco</em>R1</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
| - | An illegal | + | <img src="/wiki/images/b/bc/Kit_August22_c.jpg"> |
| + | An illegal <em>Eco</em>R1 restriction site was removed.<br> | ||
<br> | <br> | ||
<strong>PCR</strong> | <strong>PCR</strong> | ||
| Line 4,125: | Line 4,123: | ||
<tr> | <tr> | ||
<th colspan="3">Sample</th> | <th colspan="3">Sample</th> | ||
| - | <th></th> | + | <th rowspan="2">DNA polymerase</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,131: | Line 4,129: | ||
<th>Insert</th> | <th>Insert</th> | ||
<th>Primer</th> | <th>Primer</th> | ||
| - | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>か pGEX6P-2</td> | <td>か pGEX6P-2</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
| - | <td>terpinene3-prefix and | + | <td>terpinene3-prefix and terpinene3-suffix</td> |
| - | <td>KOD-FX- | + | <td>KOD-FX-NEO-Plus</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 4,146: | Line 4,143: | ||
<table class="materials"> | <table class="materials"> | ||
<tr> | <tr> | ||
| - | <th></th> | + | <th rowspan="2">Lane</th> |
<th colspan="4">Sample</th> | <th colspan="4">Sample</th> | ||
| - | <th></th> | + | <th rowspan="2">DNA polymerase</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | |||
<th>Marker</th> | <th>Marker</th> | ||
<th>Vector</th> | <th>Vector</th> | ||
<th>Insert</th> | <th>Insert</th> | ||
<th>Primer</th> | <th>Primer</th> | ||
| - | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,170: | Line 4,165: | ||
<td></td> | <td></td> | ||
<td>か pGEX6P-2</td> | <td>か pGEX6P-2</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
| - | <td>terpinene3-prefix and | + | <td>terpinene3-prefix and terpinene3-suffix</td> |
| - | <td>KOD-FX- | + | <td>KOD-FX-NEO-Plus</td> |
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
| + | <img src="/wiki/images/7/72/Kit_August22_1_c.jpg"> | ||
<br> | <br> | ||
<br> | <br> | ||
| Line 4,181: | Line 4,177: | ||
<h3>August 30, 2014</h3> | <h3>August 30, 2014</h3> | ||
| + | KOBAYASHI | ||
| + | <br> | ||
<strong>PCR</strong> | <strong>PCR</strong> | ||
<div class="m_table"> | <div class="m_table"> | ||
| Line 4,186: | Line 4,184: | ||
<tr> | <tr> | ||
<th colspan="3">Sample</th> | <th colspan="3">Sample</th> | ||
| - | <th></th> | + | <th rowspan="2">DNA polymerase</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,192: | Line 4,190: | ||
<th>Insert</th> | <th>Insert</th> | ||
<th>Primer</th> | <th>Primer</th> | ||
| - | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>か pGEX6P-2</td> | <td>か pGEX6P-2</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
| - | <td>terpinene3-prefix- | + | <td>terpinene3-prefix-<em>Xba</em>1 and terpinene3-suffix-<em>Spe</em>1</td> |
| - | <td>Taq</td> | + | <td><em>Taq</em></td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 4,207: | Line 4,204: | ||
<table class="materials"> | <table class="materials"> | ||
<tr> | <tr> | ||
| - | <th></th> | + | <th rowspan="2">Lane</th> |
<th colspan="4">Sample</th> | <th colspan="4">Sample</th> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | |||
<th>Marker</th> | <th>Marker</th> | ||
<th>Vector</th> | <th>Vector</th> | ||
| Line 4,228: | Line 4,224: | ||
<td></td> | <td></td> | ||
<td>か pGEX6P-2</td> | <td>か pGEX6P-2</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
| - | <td>terpinene3-prefix- | + | <td>terpinene3-prefix-<em>Xba</em>1 and terpinene3-suffix-<em>Spe</em>1</td> |
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
<br> | <br> | ||
| - | Made total | + | <img src="/wiki/images/3/31/Kit_August30_c.jpg"> |
| + | Made total amount of 295 μL of PCR samples. | ||
<br> | <br> | ||
</tr> | </tr> | ||
<br> | <br> | ||
</p> | </p> | ||
| + | </div> | ||
| + | </div> | ||
<div class="accordion"> | <div class="accordion"> | ||
| Line 4,246: | Line 4,245: | ||
<p class="sentence"> | <p class="sentence"> | ||
<h3>September 18, 2014</h3> | <h3>September 18, 2014</h3> | ||
| + | KOBAYASHI | ||
| + | <br> | ||
<strong>Miniprep</strong> | <strong>Miniprep</strong> | ||
<div class="m_table"> | <div class="m_table"> | ||
| Line 4,280: | Line 4,281: | ||
<tr> | <tr> | ||
<th colspan="3">Sample</th> | <th colspan="3">Sample</th> | ||
| - | <th></th> | + | <th rowspan="2">Buffer</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,286: | Line 4,287: | ||
<th>Insert</th> | <th>Insert</th> | ||
<th>Enzyme</th> | <th>Enzyme</th> | ||
| - | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,297: | Line 4,297: | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
<td>BBa_J04450</td> | <td>BBa_J04450</td> | ||
| - | <td> | + | <td><em>Xba</em>1, <em>Spe</em>1</td> |
<td>Fast Digest buffer</td> | <td>Fast Digest buffer</td> | ||
</tr> | </tr> | ||
| Line 4,314: | Line 4,314: | ||
<table class="materials"> | <table class="materials"> | ||
<tr> | <tr> | ||
| - | <th></th> | + | <th rowspan="2">Lane</th> |
<th colspan="4">Sample</th> | <th colspan="4">Sample</th> | ||
| - | <th></th> | + | <th rowspan="2">Enzyme</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | |||
<th>Marker</th> | <th>Marker</th> | ||
<th>Host</th> | <th>Host</th> | ||
<th>Vector</th> | <th>Vector</th> | ||
<th>Insert</th> | <th>Insert</th> | ||
| - | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,332: | Line 4,330: | ||
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
| - | <td>/td> | + | <td></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,340: | Line 4,338: | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
<td>BBa_J04450</td> | <td>BBa_J04450</td> | ||
| - | <td><em>Eco</em>R1, <em>Pst</em>1/td> | + | <td><em>Eco</em>R1, <em>Pst</em>1</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,348: | Line 4,346: | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
<td>BBa_J04450</td> | <td>BBa_J04450</td> | ||
| - | <td> | + | <td><em>Xba</em>1, <em>Spe</em>1</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td> | + | <td>4</td> |
<td></td> | <td></td> | ||
<td>DH5α</td> | <td>DH5α</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
<td>BBa_J04450</td> | <td>BBa_J04450</td> | ||
| - | <td>/td> | + | <td></td> |
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
| + | <img src="/wiki/images/2/29/Kit_September18_c.jpg"> | ||
<br> | <br> | ||
<strong>PCR</strong> | <strong>PCR</strong> | ||
| Line 4,366: | Line 4,365: | ||
<tr> | <tr> | ||
<th colspan="3">Sample</th> | <th colspan="3">Sample</th> | ||
| + | <th rowspan="2">DNA polymerase</th> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,371: | Line 4,371: | ||
<th>Insert</th> | <th>Insert</th> | ||
<th>Primer</th> | <th>Primer</th> | ||
| - | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>か pGEX6P-2</td> | <td>か pGEX6P-2</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
| - | <td>terpinene3-prefix- | + | <td>terpinene3-prefix-<em>Xba</em>1 and terpinene3-suffix-<em>Spe</em>1</td> |
| - | <td>Taq</td> | + | <td><em>Taq</em></td> |
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
<br> | <br> | ||
| - | <strong> | + | <strong>DNA Refinement 1</strong> |
<div class="m_table"> | <div class="m_table"> | ||
<table class="materials"> | <table class="materials"> | ||
| Line 4,393: | Line 4,392: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
| - | <td>terpinene3-prefix- | + | <td>terpinene3-prefix-<em>Xba</em>1 and terpinene3-suffix-<em>Spe</em>1</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 4,405: | Line 4,404: | ||
<tr> | <tr> | ||
<th colspan="3">Sample</th> | <th colspan="3">Sample</th> | ||
| - | <th></th> | + | <th rowspan="2">Enzyme</th> |
| - | <th></th> | + | <th rowspan="2">Buffer</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,412: | Line 4,411: | ||
<th>Insert</th> | <th>Insert</th> | ||
<th>Primer</th> | <th>Primer</th> | ||
| - | |||
| - | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,423: | Line 4,420: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td> | + | <td>か pGEX6P-2</td> |
<td>muta-CuMTS3</td> | <td>muta-CuMTS3</td> | ||
| - | <td>terpinene3-prefix- | + | <td>terpinene3-prefix-<em>Xba</em>1 and terpinene3-suffix-<em>Spe</em>1</td> |
<td><em>Xba</em>1, <em>Spe</em>1</td> | <td><em>Xba</em>1, <em>Spe</em>1</td> | ||
<td>Fast Digest</td> | <td>Fast Digest</td> | ||
| Line 4,446: | Line 4,443: | ||
<tr> | <tr> | ||
<td>muta-CuMTS3</td> | <td>muta-CuMTS3</td> | ||
| - | <td>terpinene3-prefix- | + | <td>terpinene3-prefix-<em>Xba</em>1 and terpinene3-suffix-<em>Spe</em>1</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,458: | Line 4,455: | ||
<tr> | <tr> | ||
<th colspan="3">Sample</th> | <th colspan="3">Sample</th> | ||
| - | <th></th> | + | <th rowspan="2">Enzyme</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,464: | Line 4,461: | ||
<th>DNA</th> | <th>DNA</th> | ||
<th>Primer</th> | <th>Primer</th> | ||
| - | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
<td>muta-CuMTS3</td> | <td>muta-CuMTS3</td> | ||
| - | <td>terpinene3-prefix- | + | <td>terpinene3-prefix-<em>Xba</em>1 and terpinene3-suffix-<em>Spe<em>1</td> |
<td><em>Spe</em>1, <em>Xba</em>1</td> | <td><em>Spe</em>1, <em>Xba</em>1</td> | ||
</tr> | </tr> | ||
| Line 4,476: | Line 4,472: | ||
</div> | </div> | ||
</tr> | </tr> | ||
| - | |||
DNA ligase: DNA Ligation Kit ‹Mighty Mix› (TAKARA BIO INC.) | DNA ligase: DNA Ligation Kit ‹Mighty Mix› (TAKARA BIO INC.) | ||
| + | <br> | ||
<br> | <br> | ||
<strong>Transformation</strong> | <strong>Transformation</strong> | ||
| Line 4,483: | Line 4,479: | ||
<table class="materials"> | <table class="materials"> | ||
<tr> | <tr> | ||
| - | <th colspan=" | + | <th colspan="4">Sample</th> |
| - | + | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,496: | Line 4,491: | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
<td>muta-CuMTS3</td> | <td>muta-CuMTS3</td> | ||
| - | <td>terpinene3-prefix- | + | <td>terpinene3-prefix-<em>Xba</em>1 and terpinene3-suffix-<em>Spe</em>1</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,507: | Line 4,502: | ||
<h3>September 19, 2014</h3> | <h3>September 19, 2014</h3> | ||
| + | KOBAYASHI | ||
| + | <br> | ||
<strong>Colony Isolation</strong> | <strong>Colony Isolation</strong> | ||
<br> | <br> | ||
| Line 4,515: | Line 4,512: | ||
<tr> | <tr> | ||
<th colspan="4">Sample</th> | <th colspan="4">Sample</th> | ||
| - | <th></th> | + | <th rowspan="2">Section</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,522: | Line 4,519: | ||
<th>Insert</th> | <th>Insert</th> | ||
<th>Primer</th> | <th>Primer</th> | ||
| - | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>DH5α</td> | <td>DH5α</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
| - | <td>terpinene3-prefix- | + | <td>terpinene3-prefix-<em>Xba</em>1 and terpinene3-suffix-<em>Spe</em>1</td> |
<td>A-P</td> | <td>A-P</td> | ||
</tr> | </tr> | ||
| Line 4,534: | Line 4,530: | ||
<td>DH5α</td> | <td>DH5α</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
| - | <td>terpinene3-prefix- | + | <td>terpinene3-prefix-<em>Xba</em>1 and terpinene3-suffix-<em>Spe</em>1</td> |
<td>ア-タ</td> | <td>ア-タ</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
| + | <br> | ||
<br> | <br> | ||
<strong>Colony Sweep</strong> | <strong>Colony Sweep</strong> | ||
| Line 4,557: | Line 4,554: | ||
<td>DH5α</td> | <td>DH5α</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,657: | Line 4,654: | ||
</table> | </table> | ||
</div> | </div> | ||
| + | <img src="/wiki/images/4/40/Kit_September19_c.jpg"> | ||
| + | <br> | ||
| + | Targeted colonies were identified in セ and ソ. | ||
<br> | <br> | ||
| - | |||
<br> | <br> | ||
<strong>Colony Sweep</strong> | <strong>Colony Sweep</strong> | ||
| Line 4,676: | Line 4,675: | ||
<td>DH5α</td> | <td>DH5α</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,778: | Line 4,777: | ||
<br> | <br> | ||
</tr> | </tr> | ||
| + | |||
| + | <img src="/wiki/images/e/e6/Kit_September19_1_c.jpg"> | ||
<br> | <br> | ||
Targeted colonies were identified in colony N and F. | Targeted colonies were identified in colony N and F. | ||
<br> | <br> | ||
| - | + | <br> | |
| + | Select セ, ソ, N, F | ||
| + | <br> | ||
<br> | <br> | ||
<strong>Main Culture</strong> | <strong>Main Culture</strong> | ||
| Line 4,799: | Line 4,802: | ||
<td>DH5α(N)</td> | <td>DH5α(N)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td>----------</td> | <td>----------</td> | ||
</tr> | </tr> | ||
| Line 4,805: | Line 4,808: | ||
<td>DH5α(F)</td> | <td>DH5α(F)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td>----------</td> | <td>----------</td> | ||
</tr> | </tr> | ||
| Line 4,811: | Line 4,814: | ||
<td>DH5α(セ)</td> | <td>DH5α(セ)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td>----------</td> | <td>----------</td> | ||
</tr> | </tr> | ||
| Line 4,817: | Line 4,820: | ||
<td>DH5α(ソ)</td> | <td>DH5α(ソ)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td>----------</td> | <td>----------</td> | ||
</tr> | </tr> | ||
| Line 4,823: | Line 4,826: | ||
</div> | </div> | ||
<br> | <br> | ||
| - | + | Cultivate these samples in 3mL of LB medium with ChlPhe at 30°C overnight. | |
<br> | <br> | ||
</p> | </p> | ||
| Line 4,829: | Line 4,832: | ||
<h3>September 20, 2014</h3> | <h3>September 20, 2014</h3> | ||
| + | KOBAYASHI | ||
| + | <br> | ||
<strong>Miniprep</strong> | <strong>Miniprep</strong> | ||
<br> | <br> | ||
| Line 4,844: | Line 4,849: | ||
<td>DH5α(N)</td> | <td>DH5α(N)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>DH5α(F)</td> | <td>DH5α(F)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>DH5α(セ)</td> | <td>DH5α(セ)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>DH5α(ソ)</td> | <td>DH5α(ソ)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
<br> | <br> | ||
| + | Enzyme: E/P and <em>Xba</em>1 | ||
<br> | <br> | ||
| - | |||
<br> | <br> | ||
<strong>Restriction Digest</strong> | <strong>Restriction Digest</strong> | ||
| Line 4,872: | Line 4,877: | ||
<tr> | <tr> | ||
<th colspan="4">Sample</th> | <th colspan="4">Sample</th> | ||
| - | <th>Enzyme</th> | + | <th rowspan="2">Enzyme</th> |
| - | <th>Buffer</th> | + | <th rowspan="2">Buffer</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 4,893: | Line 4,898: | ||
<td>DH5α (セ)</td> | <td>DH5α (セ)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
| Line 4,901: | Line 4,906: | ||
<td>DH5α (ソ)</td> | <td>DH5α (ソ)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
| Line 4,909: | Line 4,914: | ||
<td>DH5α (F)</td> | <td>DH5α (F)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
| Line 4,917: | Line 4,922: | ||
<td>DH5α (N)</td> | <td>DH5α (N)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
| Line 4,933: | Line 4,938: | ||
<td>DH5α (ソ)</td> | <td>DH5α (ソ)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Eco</em>R1, <em>Pst</em>1</td> | <td><em>Eco</em>R1, <em>Pst</em>1</td> | ||
<td>Buffer H</td> | <td>Buffer H</td> | ||
| Line 4,941: | Line 4,946: | ||
<td>DH5α (F)</td> | <td>DH5α (F)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Eco</em>R1, <em>Pst</em>1</td> | <td><em>Eco</em>R1, <em>Pst</em>1</td> | ||
<td>Buffer H</td> | <td>Buffer H</td> | ||
| Line 4,949: | Line 4,954: | ||
<td>DH5α (N)</td> | <td>DH5α (N)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Eco</em>R1, <em>Pst</em>1</td> | <td><em>Eco</em>R1, <em>Pst</em>1</td> | ||
<td>Buffer H</td> | <td>Buffer H</td> | ||
| Line 4,957: | Line 4,962: | ||
<td></td> | <td></td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Eco</em>R1, <em>Pst</em>1</td> | <td><em>Eco</em>R1, <em>Pst</em>1</td> | ||
<td>Buffer H</td> | <td>Buffer H</td> | ||
| Line 4,973: | Line 4,978: | ||
<td>DH5α (セ)</td> | <td>DH5α (セ)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Xba</em>1</td> | <td><em>Xba</em>1</td> | ||
<td>FD buffer</td> | <td>FD buffer</td> | ||
| Line 4,981: | Line 4,986: | ||
<td>DH5α (ソ)</td> | <td>DH5α (ソ)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Xba</em>1</td> | <td><em>Xba</em>1</td> | ||
<td>FD buffer</td> | <td>FD buffer</td> | ||
| Line 4,989: | Line 4,994: | ||
<td>DH5α (F)</td> | <td>DH5α (F)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Xba</em>1</td> | <td><em>Xba</em>1</td> | ||
<td>FD buffer</td> | <td>FD buffer</td> | ||
| Line 4,997: | Line 5,002: | ||
<td>DH5α (N)</td> | <td>DH5α (N)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Xba</em>1</td> | <td><em>Xba</em>1</td> | ||
<td>FD buffer</td> | <td>FD buffer</td> | ||
| Line 5,008: | Line 5,013: | ||
<table class="materials"> | <table class="materials"> | ||
<tr> | <tr> | ||
| - | <th>Lane</th> | + | <th rowspan="2">Lane</th> |
| - | <th | + | <th colspan="4">Sample</th> |
| - | <th>Enzyme</th> | + | <th rowspan="2">Enzyme</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 5,031: | Line 5,036: | ||
<td>DH5α (セ)</td> | <td>DH5α (セ)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td></td> | <td></td> | ||
</tr> | </tr> | ||
| Line 5,039: | Line 5,044: | ||
<td>DH5α (ソ)</td> | <td>DH5α (ソ)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td></td> | <td></td> | ||
</tr> | </tr> | ||
| Line 5,047: | Line 5,052: | ||
<td>DH5α (F)</td> | <td>DH5α (F)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td></td> | <td></td> | ||
</tr> | </tr> | ||
| Line 5,055: | Line 5,060: | ||
<td>DH5α (N)</td> | <td>DH5α (N)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td></td> | <td></td> | ||
</tr> | </tr> | ||
| Line 5,071: | Line 5,076: | ||
<td>DH5α (セ)</td> | <td>DH5α (セ)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Eco</em>R1, <em>Pst</em>1</td> | <td><em>Eco</em>R1, <em>Pst</em>1</td> | ||
</tr> | </tr> | ||
| Line 5,079: | Line 5,084: | ||
<td>DH5α (ソ)</td> | <td>DH5α (ソ)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Eco</em>R1, <em>Pst</em>1</td> | <td><em>Eco</em>R1, <em>Pst</em>1</td> | ||
</tr> | </tr> | ||
| Line 5,087: | Line 5,092: | ||
<td>DH5α (F)</td> | <td>DH5α (F)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Eco</em>R1, <em>Pst</em>1</td> | <td><em>Eco</em>R1, <em>Pst</em>1</td> | ||
</tr> | </tr> | ||
| Line 5,095: | Line 5,100: | ||
<td>DH5α (N)</td> | <td>DH5α (N)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Eco</em>R1, <em>Pst</em>1</td> | <td><em>Eco</em>R1, <em>Pst</em>1</td> | ||
</tr> | </tr> | ||
| Line 5,103: | Line 5,108: | ||
<td></td> | <td></td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Eco</em>R1, <em>Pst</em>1</td> | <td><em>Eco</em>R1, <em>Pst</em>1</td> | ||
</tr> | </tr> | ||
| Line 5,127: | Line 5,132: | ||
<td>DH5α (セ)</td> | <td>DH5α (セ)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Xba</em>1</td> | <td><em>Xba</em>1</td> | ||
</tr> | </tr> | ||
| Line 5,135: | Line 5,140: | ||
<td>DH5α (ソ)</td> | <td>DH5α (ソ)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Xba</em>1</td> | <td><em>Xba</em>1</td> | ||
</tr> | </tr> | ||
| Line 5,143: | Line 5,148: | ||
<td>DH5α (F)</td> | <td>DH5α (F)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Xba</em>1</td> | <td><em>Xba</em>1</td> | ||
</tr> | </tr> | ||
| Line 5,151: | Line 5,156: | ||
<td>DH5α (N)</td> | <td>DH5α (N)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td><em>Xba</em>1</td> | <td><em>Xba</em>1</td> | ||
</tr> | </tr> | ||
| Line 5,157: | Line 5,162: | ||
</div> | </div> | ||
<br> | <br> | ||
| - | </ | + | <img src="/wiki/images/e/e5/Kit_September20_c.jpg"> |
| - | < | + | <div class="clear"><hr /></div> |
Sample F was selected as a submission plasmid. | Sample F was selected as a submission plasmid. | ||
| + | <br> | ||
<br> | <br> | ||
<strong>Main Culture</strong> | <strong>Main Culture</strong> | ||
| Line 5,177: | Line 5,183: | ||
<td>DH5α(F)</td> | <td>DH5α(F)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td>----------</td> | <td>----------</td> | ||
</tr> | </tr> | ||
| Line 5,183: | Line 5,189: | ||
<td>DH5α(F)</td> | <td>DH5α(F)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td>----------</td> | <td>----------</td> | ||
</tr> | </tr> | ||
| Line 5,189: | Line 5,195: | ||
<td>DH5α(F)</td> | <td>DH5α(F)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td>----------</td> | <td>----------</td> | ||
</tr> | </tr> | ||
| Line 5,195: | Line 5,201: | ||
</div> | </div> | ||
<br> | <br> | ||
| - | + | Cultivate these samples in 3mL of LB medium with ChlPhe at 30°C overnight. | |
<br> | <br> | ||
</p> | </p> | ||
| Line 5,201: | Line 5,207: | ||
<h3>September 21, 2014</h3> | <h3>September 21, 2014</h3> | ||
| + | KOBAYASHI | ||
| + | <br> | ||
<strong>Miniprep</strong> | <strong>Miniprep</strong> | ||
<br> | <br> | ||
| Line 5,217: | Line 5,225: | ||
<td>DH5α(F)</td> | <td>DH5α(F)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td></td> | <td></td> | ||
</tr> | </tr> | ||
| Line 5,223: | Line 5,231: | ||
<td>DH5α(F)</td> | <td>DH5α(F)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td></td> | <td></td> | ||
</tr> | </tr> | ||
| Line 5,229: | Line 5,237: | ||
<td>DH5α(F)</td> | <td>DH5α(F)</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td></td> | <td></td> | ||
</tr> | </tr> | ||
| Line 5,241: | Line 5,249: | ||
<tr> | <tr> | ||
<th colspan="5">Sample</th> | <th colspan="5">Sample</th> | ||
| - | <th>Enzyme</th> | + | <th rowspan="2">Enzyme</th> |
| - | <th>Buffer</th> | + | <th rowspan="2">Buffer</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 5,336: | Line 5,344: | ||
<td>F</td> | <td>F</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td></td> | <td></td> | ||
<td><em>Eco</em>R1</td> | <td><em>Eco</em>R1</td> | ||
| Line 5,345: | Line 5,353: | ||
<td>F</td> | <td>F</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td></td> | <td></td> | ||
<td><em>Pst</em>1</td> | <td><em>Pst</em>1</td> | ||
| Line 5,354: | Line 5,362: | ||
<td>F</td> | <td>F</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td></td> | <td></td> | ||
<td><em>Eco</em>R1, <em>Pst</em>1</td> | <td><em>Eco</em>R1, <em>Pst</em>1</td> | ||
| Line 5,363: | Line 5,371: | ||
<td>F</td> | <td>F</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td></td> | <td></td> | ||
<td><em>Spe</em>1</td> | <td><em>Spe</em>1</td> | ||
| Line 5,372: | Line 5,380: | ||
<td>F</td> | <td>F</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td></td> | <td></td> | ||
<td><em>Xba</em>1</td> | <td><em>Xba</em>1</td> | ||
| Line 5,381: | Line 5,389: | ||
<td>F</td> | <td>F</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td></td> | <td></td> | ||
<td><em>Spe</em>1, <em>Xba</em>1</td> | <td><em>Spe</em>1, <em>Xba</em>1</td> | ||
| Line 5,390: | Line 5,398: | ||
<td>F</td> | <td>F</td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
| Line 5,402: | Line 5,410: | ||
<table class="materials"> | <table class="materials"> | ||
<tr> | <tr> | ||
| - | <th>Lane</th> | + | <th rowspan="2">Lane</th> |
| - | <th | + | <th colspan="5">Sample</th> |
| - | <th>Enzyme</th> | + | <th rowspan="2">Enzyme</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 5,411: | Line 5,419: | ||
<th>Vector</th> | <th>Vector</th> | ||
<th>Insert</th> | <th>Insert</th> | ||
| + | <th>Primer</th> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>1</td> | <td>1</td> | ||
| + | <td>1kb Ladder</td> | ||
| + | <td></td> | ||
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
| - | |||
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
| Line 5,423: | Line 5,433: | ||
<td>2</td> | <td>2</td> | ||
<td></td> | <td></td> | ||
| - | <td> | + | <td></td> |
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>BBa_J04450</td> |
<td></td> | <td></td> | ||
| + | <td><em>Eco</em>R1</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>3</td> | <td>3</td> | ||
<td></td> | <td></td> | ||
| - | <td> | + | <td></td> |
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>BBa_J04450</td> |
<td></td> | <td></td> | ||
| + | <td><em>Pst</em>1</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>4</td> | <td>4</td> | ||
<td></td> | <td></td> | ||
| - | <td> | + | <td></td> |
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>BBa_J04450</td> |
<td></td> | <td></td> | ||
| + | <td><em>Eco</em>R1, <em>Pst</em>1</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>5</td> | <td>5</td> | ||
<td></td> | <td></td> | ||
| - | <td> | + | <td></td> |
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>BBa_J04450</td> |
<td></td> | <td></td> | ||
| + | <td><em>Spe</em>1</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>6</td> | <td>6</td> | ||
| - | |||
| - | |||
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
| + | <td>pSB1C3</td> | ||
| + | <td>BBa_J04450</td> | ||
<td></td> | <td></td> | ||
| + | <td><em>Xba</em>1</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>7</td> | <td>7</td> | ||
<td></td> | <td></td> | ||
| - | <td> | + | <td></td> |
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>BBa_J04450</td> |
| - | <td><em> | + | <td></td> |
| + | <td><em>Spe</em>1, <em>Xba</em>1</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>8</td> | <td>8</td> | ||
<td></td> | <td></td> | ||
| - | <td> | + | <td></td> |
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>BBa_J04450</td> |
| - | <td> | + | <td></td> |
| + | <td></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>9</td> | <td>9</td> | ||
| + | <td>λDNA-HindⅢ</td> | ||
| + | <td></td> | ||
| + | <td></td> | ||
| + | <td></td> | ||
| + | <td></td> | ||
<td></td> | <td></td> | ||
| - | |||
| - | |||
| - | |||
| - | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>10</td> | <td>10</td> | ||
<td></td> | <td></td> | ||
| - | <td> | + | <td>F</td> |
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
| - | <td>< | + | <td></td> |
| + | <td><em>Eco</em>R1</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>11</td> | <td>11</td> | ||
<td></td> | <td></td> | ||
| - | <td></td> | + | <td>F</td> |
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
| - | <td>< | + | <td></td> |
| + | <td><em>Pst</em>1</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>12</td> | <td>12</td> | ||
| - | |||
| - | |||
| - | |||
<td></td> | <td></td> | ||
| + | <td>F</td> | ||
| + | <td>pSB1C3</td> | ||
| + | <td>muta-CuMTS3</td> | ||
<td></td> | <td></td> | ||
| + | <td><em>Eco</em>R1, <em>Pst</em>1</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>13</td> | <td>13</td> | ||
<td></td> | <td></td> | ||
| - | <td></td> | + | <td>F</td> |
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| + | <td>muta-CuMTS3</td> | ||
<td></td> | <td></td> | ||
| - | <td><em> | + | <td><em>Spe</em>1</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>14</td> | <td>14</td> | ||
<td></td> | <td></td> | ||
| - | <td> | + | <td>F</td> |
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
| + | <td></td> | ||
<td><em>Xba</em>1</td> | <td><em>Xba</em>1</td> | ||
</tr> | </tr> | ||
| Line 5,527: | Line 5,550: | ||
<td>15</td> | <td>15</td> | ||
<td></td> | <td></td> | ||
| - | <td> | + | <td>F</td> |
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
| - | <td><em>Xba</em>1</td> | + | <td></td> |
| + | <td><em>Spe</em>1, <em>Xba</em>1</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>16</td> | <td>16</td> | ||
<td></td> | <td></td> | ||
| - | <td> | + | <td>F</td> |
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td> | + | <td>muta-CuMTS3</td> |
| - | <td> | + | <td></td> |
| - | + | ||
| - | + | ||
| - | + | ||
<td></td> | <td></td> | ||
| - | |||
| - | |||
| - | |||
| - | |||
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
| - | < | + | <img src="/wiki/images/d/d7/Kit_September21_c.jpg"> |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
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| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
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| - | |||
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| - | |||
| - | |||
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| - | |||
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| - | |||
| - | |||
<br> | <br> | ||
<br></p> | <br></p> | ||
| Line 5,634: | Line 5,581: | ||
<h3>October 1, 2014</h3> | <h3>October 1, 2014</h3> | ||
| - | <strong>Protein Extraction (<em>S.cerevisiae</em>)</strong> | + | KOBAYASHI |
| + | <br> | ||
| + | <strong>Protein Extraction (<em>S. cerevisiae</em>)</strong> | ||
<br> | <br> | ||
<div class="m_table"> | <div class="m_table"> | ||
| Line 5,657: | Line 5,606: | ||
<td>p427-TEF</td> | <td>p427-TEF</td> | ||
<td>CuMTSE2</td> | <td>CuMTSE2</td> | ||
| - | <td> | + | <td>pGEX6P-2F1, pGEX6P-2R</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 5,663: | Line 5,612: | ||
<td>p427-TEF</td> | <td>p427-TEF</td> | ||
<td>CuMTS3</td> | <td>CuMTS3</td> | ||
| - | <td> | + | <td>pGEX6P-2F2, pGEX6P-2R</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 5,669: | Line 5,618: | ||
<td>p427-TEF</td> | <td>p427-TEF</td> | ||
<td>CuMTS62</td> | <td>CuMTS62</td> | ||
| - | <td> | + | <td>pGEX6P-2F2, pGEX6P-2R</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 5,700: | Line 5,649: | ||
<td>p427-TEF</td> | <td>p427-TEF</td> | ||
<td>CuMTSE2</td> | <td>CuMTSE2</td> | ||
| - | <td> | + | <td>pGEX6P-2F1, pGEX6P-2R</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 5,707: | Line 5,656: | ||
<td>p427-TEF</td> | <td>p427-TEF</td> | ||
<td>CuMTS3</td> | <td>CuMTS3</td> | ||
| - | <td> | + | <td>pGEX6P-2F2, pGEX6P-2R</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 5,714: | Line 5,663: | ||
<td>p427-TEF</td> | <td>p427-TEF</td> | ||
<td>CuMTS62</td> | <td>CuMTS62</td> | ||
| - | <td> | + | <td>pGEX6P-2F2, pGEX6P-2R</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 5,724: | Line 5,673: | ||
<h3>October 2, 2014</h3> | <h3>October 2, 2014</h3> | ||
| + | KOBAYASHI | ||
| + | <br> | ||
<strong>Preculture</strong> | <strong>Preculture</strong> | ||
<br> | <br> | ||
| Line 5,747: | Line 5,698: | ||
<td>p427-TEF</td> | <td>p427-TEF</td> | ||
<td>CuMTS3</td> | <td>CuMTS3</td> | ||
| - | <td> | + | <td>pGEX6P-2F2, pGEX6P-2R</td> |
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
| + | <br> | ||
| + | Cultivate these samples in 3mL of SD medium with G418 at 30°C overnight. | ||
<br> | <br> | ||
<br> | <br> | ||
| Line 5,778: | Line 5,731: | ||
<td>p427-TEF</td> | <td>p427-TEF</td> | ||
<td>CuMTSE2</td> | <td>CuMTSE2</td> | ||
| - | <td> | + | <td>pGEX6P-2F1, pGEX6P-2R</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 5,785: | Line 5,738: | ||
<td>p427-TEF</td> | <td>p427-TEF</td> | ||
<td>CuMTS3</td> | <td>CuMTS3</td> | ||
| - | <td> | + | <td>pGEX6P-2F2, pGEX6P-2R</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 5,792: | Line 5,745: | ||
<td>p427-TEF</td> | <td>p427-TEF</td> | ||
<td>CuMTS62</td> | <td>CuMTS62</td> | ||
| - | <td> | + | <td>pGEX6P-2F2, pGEX6P-2R</td> |
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
<br> | <br> | ||
| - | </ | + | <img src="/wiki/images/e/e0/Kit_October2_c.jpg"> |
</p> | </p> | ||
<h3>October 3, 2014</h3> | <h3>October 3, 2014</h3> | ||
| + | KOBAYASHI | ||
| + | <br> | ||
<strong>Main culture</strong> | <strong>Main culture</strong> | ||
<br> | <br> | ||
| - | + | Cultivate following samples in Big Scale with G418 at 28°C overnight in shaken culture. | |
<br> | <br> | ||
<div class="m_table"> | <div class="m_table"> | ||
| Line 5,826: | Line 5,781: | ||
<td>p427-TEF</td> | <td>p427-TEF</td> | ||
<td>CuMTS3</td> | <td>CuMTS3</td> | ||
| - | <td> | + | <td>pGEX6P-2F2, pGEX6P-2R</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 6,022: | Line 5,977: | ||
<h3>October 4, 2014</h3> | <h3>October 4, 2014</h3> | ||
| + | KOBAYASHI | ||
| + | <br> | ||
<strong>Colony Isolation</strong> | <strong>Colony Isolation</strong> | ||
<br> | <br> | ||
| Line 6,078: | Line 6,035: | ||
<td>p427-TEF</td> | <td>p427-TEF</td> | ||
<td>CuMTS3</td> | <td>CuMTS3</td> | ||
| - | <td> | + | <td>pGEX6P-2F2, pGEX6P-2R</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 6,101: | Line 6,058: | ||
<td>p427-TEF</td> | <td>p427-TEF</td> | ||
<td>CuMTSE2</td> | <td>CuMTSE2</td> | ||
| - | <td> | + | <td>pGEX6P-2F2, pGEX6P-2R</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 6,107: | Line 6,064: | ||
<td>p427-TEF</td> | <td>p427-TEF</td> | ||
<td>CuMTS62</td> | <td>CuMTS62</td> | ||
| - | <td> | + | <td>pGEX6P-2F2, pGEX6P-2R</td> |
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
<br> | <br> | ||
| - | + | Cultivate samples in 3mL of SD medium with G418 at 30°C overnight. | |
<br> | <br> | ||
</tr> | </tr> | ||
| Line 6,119: | Line 6,076: | ||
<h3>October 5, 2014</h3> | <h3>October 5, 2014</h3> | ||
| + | KOBAYASHI | ||
| + | <br> | ||
<strong>Miniprep</strong> | <strong>Miniprep</strong> | ||
<br> | <br> | ||
| Line 6,124: | Line 6,083: | ||
<table class="materials"> | <table class="materials"> | ||
<tr> | <tr> | ||
| - | <th>Host</th> | + | <th rowspan="2">Host</th> |
<th colspan="2">Sample</th> | <th colspan="2">Sample</th> | ||
</tr> | </tr> | ||
| Line 6,172: | Line 6,131: | ||
<td></td> | <td></td> | ||
<td>pSB1C3</td> | <td>pSB1C3</td> | ||
| - | <td></td> | + | <td>K1448000</td> |
<td><em>Eco</em>R1, <em>Pst</em>1</td> | <td><em>Eco</em>R1, <em>Pst</em>1</td> | ||
</tr> | </tr> | ||
| Line 6,181: | Line 6,140: | ||
</p> | </p> | ||
| + | <h3>October 8, 2014</h3> | ||
| + | KOBAYASHI | ||
<br> | <br> | ||
| + | <strong>GC-MS analysis</strong> | ||
<br> | <br> | ||
| - | < | + | <div class="m_table"> |
| - | < | + | <table class="materials"> |
| - | < | + | <tr> |
| - | < | + | <th rowspan="2">Host</th> |
| - | < | + | <th colspan="3">Sample</th> |
| - | < | + | </tr> |
| + | <tr> | ||
| + | <th>Vector</th> | ||
| + | <th>Insert</th> | ||
| + | <th>Primer</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>BY4247</td> | ||
| + | <td>p427-TEF</td> | ||
| + | <td></td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>BY4247</td> | ||
| + | <td>p427-TEF</td> | ||
| + | <td>CuMTS3</td> | ||
| + | <td>pGEX6P-2F2, pGEX6P-2R</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
<br> | <br> | ||
| - | < | + | <strong>Preculture</strong> |
| - | < | + | |
| - | + | ||
| - | + | ||
| - | + | ||
<br> | <br> | ||
| - | < | + | <div class="m_table"> |
| + | |||
| + | <table class="materials"> | ||
| + | <tr> | ||
| + | <th rowspan="2">Host</th> | ||
| + | <th colspan="3">Sample</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <th>Vector</th> | ||
| + | <th>Insert</th> | ||
| + | <th>Primer</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>BY4247</td> | ||
| + | <td>p427-TEF</td> | ||
| + | <td></td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>BY4247</td> | ||
| + | <td>p427-TEF</td> | ||
| + | <td>CuMTS3</td> | ||
| + | <td>pGEX6P-2F2, pGEX6P-2R</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
<br> | <br> | ||
| - | < | + | </tr> |
| - | < | + | </p> |
| - | < | + | |
| - | < | + | |
| - | + | <h3>October 9, 2014</h3> | |
| + | KOBAYASHI | ||
<br> | <br> | ||
| - | < | + | <strong>Main Culture</strong> |
<br> | <br> | ||
| - | < | + | <div class="m_table"> |
| - | < | + | <table class="materials"> |
| - | < | + | <tr> |
| - | < | + | <th rowspan="2">Host</th> |
| - | < | + | <th colspan="3">Sample</th> |
| + | </tr> | ||
| + | <tr> | ||
| + | <th>Vector</th> | ||
| + | <th>Insert</th> | ||
| + | <th>Primer</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>BY4247</td> | ||
| + | <td>p427-TEF</td> | ||
| + | <td></td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>BY4247</td> | ||
| + | <td>p427-TEF</td> | ||
| + | <td>CuMTS3</td> | ||
| + | <td>pGEX6P-2F2, pGEX6P-2R</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
<br> | <br> | ||
| - | < | + | </tr> |
| + | </p> | ||
| + | |||
| + | |||
| + | <h3>October 10, 2014</h3> | ||
| + | KOBAYASHI | ||
<br> | <br> | ||
| - | < | + | <strong>GC Analysis</strong> |
| - | < | + | |
| - | + | ||
| - | + | ||
| - | + | ||
<br> | <br> | ||
| + | <div class="m_table"> | ||
| + | <table class="materials"> | ||
| + | <tr> | ||
| + | <th rowspan="2">Host</th> | ||
| + | <th colspan="3">Sample</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <th>Vector</th> | ||
| + | <th>Insert</th> | ||
| + | <th>Primer</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>BY4247</td> | ||
| + | <td>p427-TEF</td> | ||
| + | <td></td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>BY4247</td> | ||
| + | <td>p427-TEF</td> | ||
| + | <td>CuMTS3</td> | ||
| + | <td>pGEX6P-2F2, pGEX6P-2R</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
<br> | <br> | ||
| - | < | + | <img src="/wiki/images/b/b1/Kit_October10_c.jpg"> |
| - | < | + | <div class="clear"><hr /></div> |
| + | For More Information: <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1448000">BBa_K1448000</a> | ||
| + | </p> | ||
</div> | </div> | ||
</div> | </div> | ||
Latest revision as of 01:13, 18 October 2014
LabNote
June
June 10, 2014
OKAMOTO
Miniprep
| Host | Sample | |
|---|---|---|
| Vector | Insert | |
| BL21(DE3) | pGEX6P-2 | CuMTSE1 |
| BL21(DE3) | pGEX6P-2 | CuMTSE2 |
| BL21(DE3) | pGEX6P-2 | CuMTS3 |
| BL21(DE3) | pGEX6P-2 | CuMTS62 |
Restriction Digest
| Host | Sample | Enzyme | Buffer | |
|---|---|---|---|---|
| Vector | Insert | |||
| BL21(DE3) | pGEX6P-2 | CuMTSE1 | EcoR1 | Buffer H |
| BL21(DE3) | pGEX6P-2 | CuMTSE2 | EcoR1 | Buffer H |
| BL21(DE3) | pGEX6P-2 | CuMTS3 | EcoR1 | Buffer H |
| BL21(DE3) | pGEX6P-2 | CuMTS62 | EcoR1 | Buffer H |
| BL21(DE3) | pGEX6P-2 | CuMTSE1 | Sal1, Not1 | Buffer H |
| BL21(DE3) | pGEX6P-2 | CuMTSE2 | Sal1, Not1 | Buffer H |
| BL21(DE3) | pGEX6P-2 | CuMTS3 | Sal1, Not1 | Buffer H |
| BL21(DE3) | pGEX6P-2 | CuMTS62 | Sal1, Not1 | Buffer H |
June 11, 2014
KOBAYASHI
AGE
| Lane | Sample | Enzyme | ||
|---|---|---|---|---|
| Marker | Vector | Insert | ||
| 1 | λDNA-HindⅢ | |||
| 2 | pGEX6P-2 | CuMTSE1 | Non Treated | |
| 3 | pGEX6P-2 | CuMTSE1 | EcoR1 | |
| 4 | pGEX6P-2 | CuMTSE1 | Sal1, Not1 | |
| 5 | pGEX6P-2 | CuMTSE2 | Non Treated | |
| 6 | pGEX6P-2 | CuMTSE2 | EcoR1 | |
| 7 | pGEX6P-2 | CuMTSE2 | Sal1, Not1 | |
| 8 | pGEX6P-2 | CuMTS3 | Non Treated | |
| 9 | pGEX6P-2 | CuMTS3 | EcoR1 | |
| 10 | pGEX6P-2 | CuMTS3 | Sal1, Not1 | |
| 11 | pGEX6P-2 | CuMTS62 | Non Treated | |
| 12 | pGEX6P-2 | CuMTS62 | EcoR1 | |
| 13 | pGEX6P-2 | CuMTS62 | Sal1, Not1 | |
June 12, 2014
KOBAYASHI
Transformation
| Host | Sample | |
|---|---|---|
| Vector | Insert | |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE1 |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE2 |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTS3 |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTS62 |
June 13, 2014
KOBAYASHI
Colony Isolation
Select 6 colonies per plate from 4 plates from June 12.
| Host | Sample | Section | |
|---|---|---|---|
| Vector | Insert | ||
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE1 | 1-a, 1-b |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE2 | 2-a, 2-b |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTS3 | 3-a, 3-b |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTS62 | 6-a, 6-b |
June 14, 2014
ONISHI
Miniprep
| Host | Sample | |
|---|---|---|
| Vector | Insert | |
| 1-a | pGEX6P-2 | CuMTSE1 |
| 1-b | pGEX6P-2 | CuMTSE1 |
| 2-a | pGEX6P-2 | CuMTSE2 |
| 2-b | pGEX6P-2 | CuMTSE2 |
| 3-a | pGEX6P-2 | CuMTS3 |
| 3-b | pGEX6P-2 | CuMTS3 |
| 6-a | pGEX6P-2 | CuMTS62 |
| 6-b | pGEX6P-2 | CuMTS62 |
Miniprep
| Host | Sample | |
|---|---|---|
| Vector | Insert | |
| BL21(DE3) | pGEX6P-2 | CuMTSE1 |
| BL21(DE3) | pGEX6P-2 | CuMTSE2 |
| BL21(DE3) | pGEX6P-2 | CuMTS3 |
| BL21(DE3) | pGEX6P-2 | CuMTS62 |
Restriction Digest
| Host | Sample | Enzyme | Buffer | |
|---|---|---|---|---|
| Vector | Insert | |||
| 1-a | pGEX6P-2 | CuMTSE1 | EcoR1 | Buffer H |
| 1-b | pGEX6P-2 | CuMTSE1 | EcoR1 | Buffer H |
| 2-a | pGEX6P-2 | CuMTSE2 | EcoR1 | Buffer H |
| 2-b | pGEX6P-2 | CuMTSE2 | EcoR1 | Buffer H |
| 3-a | pGEX6P-2 | CuMTS3 | EcoR1 | Buffer H |
| 3-b | pGEX6P-2 | CuMTS3 | EcoR1 | Buffer H |
| 6-a | pGEX6P-2 | CuMTS62 | EcoR1 | Buffer H |
| 6-b | pGEX6P-2 | CuMTS62 | EcoR1 | Buffer H |
| BL21(DE3) | pGEX6P-2 | CuMTSE1 | EcoR1 | Buffer H |
| BL21(DE3) | pGEX6P-2 | CuMTSE2 | EcoR1 | Buffer H |
| BL21(DE3) | pGEX6P-2 | CuMTS3 | EcoR1 | Buffer H |
| BL21(DE3) | pGEX6P-2 | CuMTS62 | EcoR1 | Buffer H |
AGE
| Lane | Sample | Insert | Enzyme | Buffer | ||
|---|---|---|---|---|---|---|
| Marker | Host | Vector | ||||
| 1 | 1kb Ladder | |||||
| 2 | λDNA-HindⅢ | |||||
| 3 | 1-a | pGEX6P-2 | CuMTSE1 | EcoR1 | Buffer H | |
| 4 | 1-b | pGEX6P-2 | CuMTSE1 | EcoR1 | Buffer H | |
| 5 | 2-a | pGEX6P-2 | CuMTSE2 | EcoR1 | Buffer H | |
| 6 | 2-b | pGEX6P-2 | CuMTSE2 | EcoR1 | Buffer H | |
| 7 | 3-a | pGEX6P-2 | CuMTS3 | EcoR1 | Buffer H | |
| 8 | 3-b | pGEX6P-2 | CuMTS3 | EcoR1 | Buffer H | 9 | 6-a | pGEX6P-2 | CuMTS62 | EcoR1 | Buffer H |
| 10 | 6-b | pGEX6P-2 | CuMTS62 | EcoR1 | Buffer H | |
| 11 | BL21(DE3) | pGEX6P-2 | CuMTSE1 | EcoR1 | Buffer H | |
| 12 | BL21(DE3) | pGEX6P-2 | CuMTSE2 | EcoR1 | Buffer H | |
| 13 | BL21(DE3) | pGEX6P-2 | CuMTS3 | EcoR1 | Buffer H | |
| 14 | BL21(DE3) | pGEX6P-2 | CuMTS62 | EcoR1 | Buffer H | |
Apply λDNA-HindⅢ to compare and contrast AGE of July 14 and June 11.
June 16, 2014
YAMAJI
Preculture
| Host | Sample | |
|---|---|---|
| Vector | Insert | |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE1 |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE2 |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTS3 |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTS62 |
| BL21(DE3)pLysS | pGEX6P-2 | |
Main Culture
| Host | Sample | IPTG | |
|---|---|---|---|
| Vector | Insert | ||
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE1 | Induced |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE2 | Induced |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTS3 | Induced |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTS62 | Induced |
| BL21(DE3)pLysS | pGEX6P-2 | Induced | |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE1 | Non-induced |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE2 | Non-induced |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTS3 | Non-induced |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTS62 | Non-induced |
| BL21(DE3)pLysS | pGEX6P-2 | Non-induced | |
June 18, 2014
YAMAJI
Preculture
| Host | Sample | |
|---|---|---|
| Vector | Insert | |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE1 | BL21(DE3)pLysS | pGEX6P-2 | CuMTSE2 |
Main Culture
| Host | Sample | IPTG | |
|---|---|---|---|
| Vector | Insert | ||
| BL21(DE3)pLysS | pGEX6P-2 | Induced | |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE1 | Induced |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE2 | Induced |
| BL21(DE3)pLysS | pGEX6P-2 | Non-induced | |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE1 | Non-induced |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE2 | Non-induced |
Protein Extraction (E. coli)
| Host | Sample | IPTG | |
|---|---|---|---|
| Vector | Insert | ||
| BL21(DE3)pLysS | pGEX6P-2 | Induced | |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE1 | Induced |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE2 | Induced |
| BL21(DE3)pLysS | pGEX6P-2 | Non-induced | |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE1 | Non-induced |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE2 | Non-induced |
Preparations for SDS-PAGE
Add SDS sample buffer to protein extractions in a flask, incubate at 37℃ for 15 minutes and keep it at 4℃.
June 19, 2014
KITOMI
SDS-PAGE
| Host | Sample | IPTG | |
|---|---|---|---|
| Vector | Insert | ||
| BL21(DE3)pLysS | pGEX6P-2 | Induced | |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE1 | Induced |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE2 | Induced |
| BL21(DE3)pLysS | pGEX6P-2 | Non-induced | |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE1 | Non-induced |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE2 | Non-induced |
June 21, 2014
SOGA
Miniprep
| Host | Sample | |
|---|---|---|
| Vector | Insert | |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE1 |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE2 |
PCR
| Vector | Sample | DNA Polymerase | |
|---|---|---|---|
| Insert | Primer | ||
| pGEX6P-2 | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | KOD-FX-NEO |
| pGEX6P-2 | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | KOD-FX-NEO |
| pGEX6P-2 | CuMTSE2 | pGEX6P-2F1, pGEX6P-2R | KOD-FX-NEO |
| pGEX6P-2 | CuMTSE2 | pGEX6P-2F2, pGEX6P-2R | KOD-FX-NEO |
AGE
| Lane | Sample | Insert | Primer | ||
|---|---|---|---|---|---|
| Marker | Host | Vector | |||
| 1 | 1kb Ladder | ||||
| 2 | BY4247 | p427-TEF | |||
| 3 | pGEX6P-2 | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | ||
| 4 | pGEX6P-2 | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | ||
| 5 | pGEX6P-2 | CuMTSE2 | pGEX6P-2F1, pGEX6P-2R | ||
| 6 | pGEX6P-2 | CuMTSE2 | pGEX6P-2F2, pGEX6P-2R | ||
June 24, 2014
SOGA
Preculture
| Host | Sample | |
|---|---|---|
| Vector | Insert | |
| BL21(DE3)pLysS | ||
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE1 |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE2 |
June 25, 2014
KOBAYASHI
Cultivate following samples in Big Scale at 30℃ overnight in shaken culture.
| Host | Sample | |
|---|---|---|
| Vector | Insert | |
| BL21(DE3)pLysS | ||
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE1 |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE2 |
DNA Refinement 1
| Sample | |
|---|---|
| DNA | Primer |
| CuMTSE1 | pGEX6P-2F1, pGEX6P-2R |
| CuMTSE1 | pGEX6P-2F2, pGEX6P-2R |
Restriction Digest
| Vector | Sample | Enzyme | Buffer | |
|---|---|---|---|---|
| DNA | Primer | |||
| CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 | Buffer H | |
| CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | Spe1, Xho1 | Buffer H | |
| p427-TEF | Spe1, Xho1 | Buffer H | ||
DNA Refinement 2
| Vector | Sample | Enzyme | |
|---|---|---|---|
| DNA | Primer | ||
| CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 | |
| CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | Spe1, Xho1 | |
| p427-TEF | Spe1, Xho1 | ||
Main Culture
| Host | Sample | IPTG | |
|---|---|---|---|
| Vector | Insert | ||
| BL21(DE3)pLysS | pGEX6P-2 | Induced | |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE1 | Induced |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE2 | Induced |
| BL21(DE3)pLysS | pGEX6P-2 | Non-induced | |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE1 | Non-induced |
| BL21(DE3)pLysS | pGEX6P-2 | CuMTSE2 | Non-induced |
Ligation
| Sample | ||
|---|---|---|
| Vector | DNA | Primer |
| p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R |
| p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R |
| p427-TEF | CuMTSE2 | pGEX6P-2F1, pGEX6P-2R |
| p427-TEF | CuMTSE2 | pGEX6P-2F2, pGEX6P-2R |
Transformation
| Host | Sample | ||
|---|---|---|---|
| Vector | Insert | Primer | |
| DH5α | p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R |
| DH5α | p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R |
| DH5α | p427-TEF | CuMTSE2 | pGEX6P-2F1, pGEX6P-2R |
| DH5α | p427-TEF | CuMTSE2 | pGEX6P-2F2, pGEX6P-2R |
June 26, 2014
SAWANO
Colony Isolation
Isolate each colony from the plates which were inoculated on June 25.
| Host | Sample | Section | ||
|---|---|---|---|---|
| Vector | Insert | Primer | ||
| DH5α | p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | 1～4 |
| DH5α | p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | a～h |
| DH5α | p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | ア～タ |
June 27, 2014
SHIMADA
Colony Sweep
| Lane | Sample | ||
|---|---|---|---|
| Host | Vector | Insert | |
| 1 | BL21(DE3)pLysS | pGEX6P-2 | CuMTSE1 |
| 2 | 1 | ||
| 3 | 2 | ||
| 4 | 3 | ||
| 5 | 4 | ||
| 6 | a | ||
| 7 | b | ||
| 8 | c | ||
| 9 | d | ||
| 10 | e | ||
| 11 | f | ||
| 12 | g | ||
| 13 | h | ||
Colony Sweep
| Lane | Sample | ||
|---|---|---|---|
| Host | Vector | Insert | |
| 1 | BL21(DE3)pLysS | pGEX6P-2 | CuMTSE1 |
| 2 | ア | ||
| 3 | イ | ||
| 4 | ウ | ||
| 5 | エ | ||
| 6 | オ | ||
| 7 | カ | ||
| 8 | キ | ||
| 9 | ク | ||
| 10 | ケ | ||
| 11 | コ | ||
| 12 | サ | ||
| 13 | シ | ||
| 14 | ス | ||
| 15 | セ | ||
| 16 | ソ | ||
| 17 | タ | ||
Identified targeted genes in 4, c, h, イ, カ, セ
Miniprep
| Sample | |
|---|---|
| Host | |
| 4 | |
| C | |
| H | |
| イ | |
| カ | |
| セ | |
Restriction Digest
| Sample | Enzyme | Buffer |
|---|---|---|
| Host | ||
| 4 | Spe1, Xho1 | Buffer H |
| C | Spe1, Xho1 | Buffer H |
| H | Spe1, Xho1 | Buffer H |
| イ | Spe1, Xho1 | Buffer H |
| カ | Spe1, Xho1 | Buffer H |
AGE
| Lane | Sample | Primer | Enzyme | ||
|---|---|---|---|---|---|
| Marker | Host | Insert | |||
| 1 | 1kb Ladder | ||||
| 2 | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | |||
| 3 | 4 | Spe1, Xho1 | |||
| 4 | 4 | Spe1, Xho1 | |||
| 5 | 4 | Non Treated | |||
| 6 | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | |||
| 7 | c | Spe1, Xho1 | |||
| 8 | c | Spe1, Xho1 | |||
| 9 | c | Non Treated | |||
| 10 | h | Spe1, Xho1 | |||
| 11 | h | Spe1, Xho1 | |||
| 12 | h | Non Treated | |||
Transformation (electroportation)
| Host | Sample | ||
|---|---|---|---|
| Vector | Insert | Primer | |
| BY4247 | p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R |
| BY4247 | p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R |
June 30, 2014
SOGA
Inoculation
Divide each of 2 plates into 8 sections and inoculate following samples on them.
| Host | Sample | Section | ||
|---|---|---|---|---|
| Vector | Insert | Primer | ||
| BY4247 | p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | A～H |
| BY4247 | p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | 一～ハ |
July
July 1, 2014
KOBAYASHI
Protein Extraction (S. cerevisiae)
| Host | Sample | ||
|---|---|---|---|
| Vector | Insert | Primer | |
| BY4247 | p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R |
| BY4247 | p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R |
July 2, 2014
ONISHI
Miniprep
| Host | Sample | ||
|---|---|---|---|
| Vector | Insert | Primer | |
| BY4247 | p427-TEF | ||
| BY4247 | p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R |
| BY4247 | p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R |
Restriction Digest
| Sample | Enzyme | Buffer | ||
|---|---|---|---|---|
| Vector | Insert | Primer | ||
| p427-TEF | CuMTSE1 | EcoR1 | Buffer H | |
| p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | EcoR1 | Buffer H |
| p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | EcoR1 | Buffer H |
| p427-TEF | CuMTSE1 | Sac1, Kpn1 | Buffer L | |
| p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | Sac1, Kpn1 | Buffer L |
| p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | Sac1, Kpn1 | Buffer L |
AGE
| Lane | Sample | Enzyme | |||
|---|---|---|---|---|---|
| Marker | Vector | Insert | Primer | ||
| 1 | p427-TEF | Non Treated | |||
| 2 | p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | Non Treated | |
| 3 | p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | Non Treated | |
| 4 | 1kb Ladder | ||||
| 5 | p427-TEF | EcoR1 | |||
| 6 | p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | EcoR1 | |
| 7 | p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | EcoR1 | |
| 8 | 1kb Ladder | ||||
| 9 | p427-TEF | Kpn1 | |||
| 10 | p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | Kpn1 | |
| 11 | p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | Kpn1 | |
| 12 | 1kb Ladder | ||||
| 13 | p427-TEF | Sac1 | |||
| 14 | p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | Sac1 | |
| 15 | p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | Sac1 | |
Restriction Digest
| Sample | Enzyme | Buffer | ||
|---|---|---|---|---|
| Vector | Insert | Primer | ||
| p427-TEF | CuMTSE1 | EcoR1 | Buffer H | |
| p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | EcoR1 | Buffer H |
| p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | EcoR1 | Buffer H |
| p427-TEF | CuMTSE1 | Sac1 | Buffer L | |
| p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | Sac1 | Buffer L |
| p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | Sac1 | Buffer L |
| p427-TEF | CuMTSE1 | Kpn1 | Buffer L | |
| p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | Kpn1 | Buffer L |
| p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | Kpn1 | Buffer L |
| p427-TEF | CuMTSE1 | Spe1, Xho1 | Buffer H | |
| p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 | Buffer H |
| p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | Spe1, Xho1 | Buffer H |
AGE
| Lane | Sample | Enzyme | |||
|---|---|---|---|---|---|
| Marker | Vector | Insert | Primer | ||
| 1 | 1kb Ladder | ||||
| 2 | p427-TEF | CuMTSE1 | EcoR1 | ||
| 3 | p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | EcoR1 | |
| 4 | p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | EcoR1 | |
| 5 | p427-TEF | CuMTSE1 | Kpn1 | ||
| 6 | p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | Kpn1 | |
| 7 | p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | Kpn1 | |
| 8 | p427-TEF | CuMTSE1 | Sac1 | ||
| 9 | p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | Sac1 | |
| 10 | p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | Sac1 | |
| 11 | p427-TEF | CuMTSE1 | Spe1, Xho1 | ||
| 12 | p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 | |
| 13 | p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R | Spe1, Xho1 | |
July 3, 2014
OKAMOTO
PCR
| Sample | DNA Polymerase | ||
|---|---|---|---|
| Vector | Insert | Primer | |
| pGEX6P-2 | CuMTSE2 | pGEX6P-2F1, pGEX6P-2R | KOD-FX-NEO |
| pGEX6P-2 | CuMTSE2 | pGEX6P-2F2, pGEX6P-2R | KOD-FX-NEO |
| pGEX6P-2 | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | KOD-FX-NEO |
| pGEX6P-2 | CuMTS3 | pGEX6P-2F2, pGEX6P-2R | KOD-FX-NEO |
| pGEX6P-2 | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | KOD-FX-NEO |
| pGEX6P-2 | CuMTS62 | pGEX6P-2F2, pGEX6P-2R | KOD-FX-NEO |
Protein Extraction (S. cerevisiae)
| Host | Sample | ||
|---|---|---|---|
| Vector | Insert | Primer | |
| BY4247 | p427-TEF | ||
| A | p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R |
| 六 | p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R |
July 4, 2014
SHIMADA
AGE
| Lane | Sample/Marker | |||
|---|---|---|---|---|
| Marker | Vector | Insert | Primer | |
| 1 | 1kb Ladder | |||
| 2 | pGEX6P-2 | CuMTSE2 | pGEX6P-2F1, pGEX6P-2R | |
| 3 | pGEX6P-2 | CuMTSE2 | pGEX6P-2F2, pGEX6P-2R | |
| 4 | pGEX6P-2 | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | |
| 5 | pGEX6P-2 | CuMTS3 | pGEX6P-2F2, pGEX6P-2R | |
| 6 | pGEX6P-2 | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | |
| 7 | pGEX6P-2 | CuMTS62 | pGEX6P-2F2, pGEX6P-2R | |
PCR
| Sample | DNA Polymerase | ||
|---|---|---|---|
| Vector | Insert | Primer | |
| pGEX6P-2 | CuMTSE2 | pGEX6P-2F1, pGEX6P-2R | KOD-FX-NEO |
| pGEX6P-2 | CuMTSE2 | pGEX6P-2F2, pGEX6P-2R | KOD-FX-NEO |
| pGEX6P-2 | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | KOD-FX-NEO |
| pGEX6P-2 | CuMTS3 | pGEX6P-2F2, pGEX6P-2R | KOD-FX-NEO |
| pGEX6P-2 | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | KOD-FX-NEO |
| pGEX6P-2 | CuMTS62 | pGEX6P-2F2, pGEX6P-2R | KOD-FX-NEO |
AGE
| Lane | Sample | |||
|---|---|---|---|---|
| Marker | Vector | Insert | Primer | |
| 1 | 1kb Ladder | |||
| 2 | pGEX6P-2 | CuMTSE2 | pGEX6P-2F1, pGEX6P-2R | |
| 3 | pGEX6P-2 | 1kb Ladder+K83:N91 | pGEX6P-2F2, pGEX6P-2R | |
| 4 | pGEX6P-2 | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | |
| 5 | pGEX6P-2 | CuMTS3 | pGEX6P-2F2, pGEX6P-2R | |
| 6 | pGEX6P-2 | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | |
| 7 | pGEX6P-2 | CuMTS62 | pGEX6P-2F2, pGEX6P-2R | |
July 5, 2014
KOBAYASHI
SDS-PAGE
| Host | Sample | ||
|---|---|---|---|
| Vector | Insert | Primer | |
| BL21(DE3)pLysS | pGEX6P-2 | ||
| 4 | pGEX6P-2 | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R |
| C | pGEX6P-2 | CuMTSE2 | pGEX6P-2F2, pGEX6P-2R |
| BY4247 | p427-TEF | ||
| A | p427-TEF | CuMTSE1 | pGEX6P-2F1, pGEX6P-2R |
| 六 | p427-TEF | CuMTSE1 | pGEX6P-2F2, pGEX6P-2R |
July 6, 2014
SOGA
DNA Refinement 1
| Sample | |
|---|---|
| DNA | Primer |
| CuMTSE2 | pGEX6P-2F1, pGEX6P-2R |
| CuMTSE2 | pGEX6P-2F2, pGEX6P-2R |
| CuMTS3 | pGEX6P-2F1, pGEX6P-2R |
| CuMTS62 | pGEX6P-2F1, pGEX6P-2R |
Restriction Digest
| Vector | Sample | Enzyme | Buffer | |
|---|---|---|---|---|
| DNA | Primer | |||
| CuMTSE2 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 | Fast digest buffer | |
| CuMTSE2 | pGEX6P-2F2, pGEX6P-2R | Spe1, Xho1 | Fast digest buffer | |
| CuMTS3 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 | Fast digest buffer | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 | Fast digest buffer | p427-TEF | Spe1, Xho1 | Fast digest buffer |
DNA Refinement 2
| Sample | Enzyme | |
|---|---|---|
| DNA | Primer | |
| CuMTSE2 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 |
| CuMTSE2 | pGEX6P-2F2, pGEX6P-2R | Spe1, Xho1 |
Ligation
| Vector | Sample | Enzyme | |
|---|---|---|---|
| DNA | Primer | ||
| p427-TEF | CuMTSE2 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 |
| p427-TEF | CuMTSE2 | pGEX6P-2F2, pGEX6P-2R | Spe1, Xho1 |
DNA ligase: DNA Ligation Kit ‹Mighty Mix› (TAKARA BIO INC.)
Transformation (E. coli)
| Host | Sample | Enzyme | ||
|---|---|---|---|---|
| Vector | Insert | Primer | ||
| DH5α | p427-TEF | CuMTSE2 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 |
| DH5α | p427-TEF | CuMTSE2 | pGEX6P-2F2, pGEX6P-2R | Spe1, Xho1 |
| DH5α | p427-TEF | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 |
| DH5α | p427-TEF | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 |
July 7, 2014
KOBAYASHI
Colony Isolation
Isolate each colony from the plates which were inoculated on July 6.
| Host | Sample | Section | ||
|---|---|---|---|---|
| Vector | Insert | Primer | ||
| DH5α | p427-TEF | CuMTSE2 | pGEX6P-2F1, pGEX6P-2R | 1～16 |
| DH5α | p427-TEF | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | ア～エ |
| DH5α | p427-TEF | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | a～p |
Transformation (E. coli)
| Host | Sample | Enzyme | ||
|---|---|---|---|---|
| Vector | Insert | Primer | ||
| DH5α | p427-TEF | CuMTSE2 | pGEX6P-2F2, pGEX6P-2R | Spe1, Xho1 |
July 8, 2014
OKAMOTO
Colony Sweep
| Lane | Sample | |
|---|---|---|
| Host | ||
| 1 | DH5α | p427-TEF |
| 2 | a | |
| 3 | b | |
| 4 | c | |
| 5 | d | |
| 6 | e | |
| 7 | f | |
| 8 | g | |
| 9 | h | |
| 10 | i | |
| 11 | j | |
| 12 | k | |
| 13 | l | |
| 14 | m | |
| 15 | n | |
| 16 | o | |
| 17 | p | |
Colony Sweep
| Lane | Sample | |
|---|---|---|
| Host | ||
| 1 | DH5α | p427-TEF |
| 2 | ア | |
| 3 | イ | |
| 4 | ウ | |
| 5 | エ | |
Colony Sweep
| Lane | Sample | |
|---|---|---|
| Host | ||
| 1 | DH5α | p427-TEF |
| 2 | 1 | |
| 3 | 2 | |
| 4 | 3 | |
| 5 | 4 | |
| 6 | 5 | |
| 7 | 6 | |
| 8 | 7 | |
| 9 | 8 | |
| 10 | 9 | |
| 11 | 10 | |
| 12 | 11 | |
| 13 | 12 | |
| 14 | 13 | |
| 15 | 14 | |
| 16 | 15 | |
| 17 | 16 | |
July 9, 2014
OKAMOTO
Main Culture
| Sample |
|---|
| Host |
| d |
| h |
| l |
| ア |
| イ |
| エ |
Mini Prep
| Sample |
|---|
| Host |
| D |
| H |
| L |
| ア |
| イ |
| エ |
Restriction Digest
| Vector | Sample | Enzyme | Buffer | |
|---|---|---|---|---|
| Insert | Primer | |||
| p427-TEF(d) | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 | Fast digest buffer |
| p427-TEF(h) | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 | Fast digest buffer |
| p427-TEF(l) | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 | Fast digest buffer |
| p427-TEF(ア) | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 | Fast digest buffer |
| p427-TEF(イ) | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 | Fast digest buffer |
| p427-TEF(エ) | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 | Fast digest buffer |
AGE
| Lane | Sample | Enzyme | ||
|---|---|---|---|---|
| Vector | Insert | Primer | ||
| 1 | p427-TEF(ア) | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | Non Treated |
| 2 | p427-TEF(ア) | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 |
| 3 | p427-TEF(イ) | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | Non Treated |
| 4 | p427-TEF(イ) | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 |
| 5 | p427-TEF(エ) | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | Non Treated |
| 6 | p427-TEF(エ) | CuMTS3 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 |
| 7 | p427-TEF(d) | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | Non Treated |
| 8 | p427-TEF(d) | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 |
| 9 | p427-TEF(h) | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | Non Treated |
| 10 | p427-TEF(h) | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 |
| 11 | p427-TEF(l) | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | Non Treated |
| 12 | p427-TEF(l) | CuMTS62 | pGEX6P-2F1, pGEX6P-2R | Spe1, Xho1 |
August
August 19, 2014
KOBAYASHI
Conduct mutagenesis to remove an illegal restriction site (EcoR1)
InversePCR
| Sample | |||
|---|---|---|---|
| Vector | Insert | Primer | DNA polymerase |
| pGEX6P-2 | CuMTS3 | Terpinene3-mutagenesis-FP, Terpinene3-mutagenesis-RP | KOD-Plus |
August 20, 2014
KOBAYASHI
Restart mutagenesis from KOD -Plus- Mutagenesis Kit protocol No.3 to obtain plasmid (muta-CuMTS3 in pGEX-6P-2)
Transformation
| Sample | |||
|---|---|---|---|
| Host | Vector | Insert | Primer |
| DH5α | pGEX6P-2 | muta-CuMTS3 | Terpinene3-mutagenesis-FP and Terpinene3-mutagenesis-RP |
August 21, 2014
KOBAYASHI
Colony Isolation
Isolate each colony from two plates which were inoculated overnight
| Sample | Section | |||
|---|---|---|---|---|
| Host | Vector | Insert | Primer | |
| DH5α | pSB1C3 | muta-CuMTS3 | Terpinene3-mutagenesis-FP, Terpinene3-mutagenesis-RP |
あ-み |
August 22, 2014
KOBAYASHI
Restriction Digest
| Sample | Enzyme | Buffer | ||
|---|---|---|---|---|
| Host | Vector | Insert | ||
| DH5α あ | pGEX6P-2 | muta-CuMTS3 | Sal1 | Fast Digest buffer |
| DH5α か | pGEX6P-2 | muta-CuMTS3 | Sal1 | Fast Digest buffer |
| DH5α さ | pGEX6P-2 | muta-CuMTS3 | Sal1 | Fast Digest buffer |
| DH5α た | pGEX6P-2 | muta-CuMTS3 | Sal1 | Fast Digest buffer |
AGE
| Lane | Sample | Enzyme | |||
|---|---|---|---|---|---|
| Marker | Host | Vector | Insert | ||
| 1 | 1kb Ladder | ||||
| 2 | DH5α あ | pGEX6P-2 | muta-CuMTS3 | Sal1 | |
| 3 | DH5α か | pGEX6P-2 | muta-CuMTS3 | Sal1 | |
| 4 | DH5α さ | pGEX6P-2 | muta-CuMTS3 | Sal1 | |
| 5 | DH5α た | pGEX6P-2 | muta-CuMTS3 | Sal1 | |
Restriction Digest
| Sample | Enzyme | Buffer | ||
|---|---|---|---|---|
| Host | Vector | Insert | ||
| DH5α か | pGEX6P-2 | CuMTS3 | EcoR1 | Fast Digest buffer |
| DH5α か | pGEX6P-2 | muta-CuMTS3 | EcoR1 | Fast Digest buffer |
AGE
| Lane | Sample | Enzyme | |||
|---|---|---|---|---|---|
| Marker | Host | Vector | Insert | ||
| 1 | 1kb Ladder | ||||
| 2 | DH5α か | pGEX6P-2 | CuMTS3 | EcoR1 | |
| 3 | DH5α か | pGEX6P-2 | muta-CuMTS3 | EcoR1 | |
An illegal EcoR1 restriction site was removed.PCR
| Sample | DNA polymerase | ||
|---|---|---|---|
| Vector | Insert | Primer | |
| か pGEX6P-2 | muta-CuMTS3 | terpinene3-prefix and terpinene3-suffix | KOD-FX-NEO-Plus |
AGE
| Lane | Sample | DNA polymerase | |||
|---|---|---|---|---|---|
| Marker | Vector | Insert | Primer | ||
| 1 | 1kb Ladder | ||||
| 2 | か pGEX6P-2 | muta-CuMTS3 | terpinene3-prefix and terpinene3-suffix | KOD-FX-NEO-Plus | |
August 30, 2014
KOBAYASHIPCR
| Sample | DNA polymerase | ||
|---|---|---|---|
| Vector | Insert | Primer | |
| か pGEX6P-2 | muta-CuMTS3 | terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1 | Taq |
AGE
| Lane | Sample | |||
|---|---|---|---|---|
| Marker | Vector | Insert | Primer | |
| 1 | 1kb Ladder | |||
| 2 | か pGEX6P-2 | muta-CuMTS3 | terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1 | |
Made total amount of 295 μL of PCR samples.
September
September 18, 2014
KOBAYASHIMiniprep
| Sample | ||
|---|---|---|
| Host | Vector | Insert |
| DH5α | pSB1C3 | BBa_J04450 |
| DH5α | pSB1C3 | BBa_J04450 |
| DH5α | pSB1C3 | BBa_J04450 |
Restriction Digest
| Sample | Buffer | ||
|---|---|---|---|
| Vector | Insert | Enzyme | |
| pSB1C3 | BBa_J04450 | EcoR1, Pst1 | Buffer H |
| pSB1C3 | BBa_J04450 | Xba1, Spe1 | Fast Digest buffer |
| pSB1C3 | BBa_J04450 | ||
AGE
| Lane | Sample | Enzyme | |||
|---|---|---|---|---|---|
| Marker | Host | Vector | Insert | ||
| 1 | 1kb Ladder | ||||
| 2 | DH5α | pSB1C3 | BBa_J04450 | EcoR1, Pst1 | |
| 3 | DH5α | pSB1C3 | BBa_J04450 | Xba1, Spe1 | |
| 4 | DH5α | pSB1C3 | BBa_J04450 | ||
PCR
| Sample | DNA polymerase | ||
|---|---|---|---|
| Vector | Insert | Primer | |
| か pGEX6P-2 | muta-CuMTS3 | terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1 | Taq |
DNA Refinement 1
| Sample | |
|---|---|
| Insert | Primer |
| muta-CuMTS3 | terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1 |
Restriction Digest
| Sample | Enzyme | Buffer | ||
|---|---|---|---|---|
| Vector | Insert | Primer | ||
| pSB1C3 | Xba1, Spe1 | Fast Digest | ||
| か pGEX6P-2 | muta-CuMTS3 | terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1 | Xba1, Spe1 | Fast Digest |
DNA Refinement 2
| Sample | |
|---|---|
| Insert | Primer |
| muta-CuMTS3 | terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1 |
Ligation
| Sample | Enzyme | ||
|---|---|---|---|
| Vector | DNA | Primer | |
| pSB1C3 | muta-CuMTS3 | terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1 | Spe1, Xba1 |
Transformation
| Sample | |||
|---|---|---|---|
| Host | Vector | Insert | Primer |
| DH5α | pSB1C3 | muta-CuMTS3 | terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1 |
September 19, 2014
KOBAYASHIColony Isolation
Isolate each colony from the plates which were inoculated overnight.
| Sample | Section | |||
|---|---|---|---|---|
| Host | Vector | Insert | Primer | |
| DH5α | pSB1C3 | muta-CuMTS3 | terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1 | A-P |
| DH5α | pSB1C3 | muta-CuMTS3 | terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1 | ア-タ |
Colony Sweep
| Lane | Sample | ||
|---|---|---|---|
| Host | Vector | Insert | |
| 1 | DH5α | pSB1C3 | muta-CuMTS3 |
| 2 | ア | ||
| 3 | イ | ||
| 4 | ウ | ||
| 5 | エ | ||
| 6 | オ | ||
| 7 | カ | ||
| 8 | キ | ||
| 9 | ク | ||
| 10 | ケ | ||
| 11 | コ | ||
| 12 | サ | ||
| 13 | シ | ||
| 14 | ス | ||
| 15 | セ | ||
| 16 | ソ | ||
| 17 | タ | ||
Targeted colonies were identified in セ and ソ.
Colony Sweep
| Lane | Sample | ||
|---|---|---|---|
| Host | Vector | Insert | |
| 1 | DH5α | pSB1C3 | muta-CuMTS3 |
| 2 | A | ||
| 3 | B | ||
| 4 | C | ||
| 5 | D | ||
| 6 | E | ||
| 7 | F | ||
| 8 | G | ||
| 9 | H | ||
| 10 | I | ||
| 11 | J | ||
| 12 | K | ||
| 13 | L | ||
| 14 | M | ||
| 15 | N | ||
| 16 | O | ||
| 17 | P | ||
Targeted colonies were identified in colony N and F.
Select セ, ソ, N, F
Main Culture
| Sample | |||
|---|---|---|---|
| Host | Vector | Insert | Primer |
| DH5α(N) | pSB1C3 | muta-CuMTS3 | ---------- |
| DH5α(F) | pSB1C3 | muta-CuMTS3 | ---------- |
| DH5α(セ) | pSB1C3 | muta-CuMTS3 | ---------- |
| DH5α(ソ) | pSB1C3 | muta-CuMTS3 | ---------- |
Cultivate these samples in 3mL of LB medium with ChlPhe at 30°C overnight.
September 20, 2014
KOBAYASHIMiniprep
| Sample | ||
|---|---|---|
| Host | Vector | Primer |
| DH5α(N) | pSB1C3 | muta-CuMTS3 |
| DH5α(F) | pSB1C3 | muta-CuMTS3 |
| DH5α(セ) | pSB1C3 | muta-CuMTS3 |
| DH5α(ソ) | pSB1C3 | muta-CuMTS3 |
Enzyme: E/P and Xba1
Restriction Digest
| Sample | Enzyme | Buffer | |||
|---|---|---|---|---|---|
| Marker | Host | Vector | Insert | ||
| pSB1C3 | |||||
| DH5α (セ) | pSB1C3 | muta-CuMTS3 | |||
| DH5α (ソ) | pSB1C3 | muta-CuMTS3 | |||
| DH5α (F) | pSB1C3 | muta-CuMTS3 | |||
| DH5α (N) | pSB1C3 | muta-CuMTS3 | |||
| DH5α (セ) | pSB1C3 | EcoR1, Pst1 | Buffer H | ||
| DH5α (ソ) | pSB1C3 | muta-CuMTS3 | EcoR1, Pst1 | Buffer H | |
| DH5α (F) | pSB1C3 | muta-CuMTS3 | EcoR1, Pst1 | Buffer H | |
| DH5α (N) | pSB1C3 | muta-CuMTS3 | EcoR1, Pst1 | Buffer H | |
| pSB1C3 | muta-CuMTS3 | EcoR1, Pst1 | Buffer H | ||
| pSB1C3 | Xba1 | FD buffer | |||
| DH5α (セ) | pSB1C3 | muta-CuMTS3 | Xba1 | FD buffer | |
| DH5α (ソ) | pSB1C3 | muta-CuMTS3 | Xba1 | FD buffer | |
| DH5α (F) | pSB1C3 | muta-CuMTS3 | Xba1 | FD buffer | |
| DH5α (N) | pSB1C3 | muta-CuMTS3 | Xba1 | FD buffer | |
AGE
| Lane | Sample | Enzyme | |||
|---|---|---|---|---|---|
| Marker | Host | Vector | Insert | ||
| 1 | pSB1C3 | ||||
| 2 | DH5α (セ) | pSB1C3 | muta-CuMTS3 | ||
| 3 | DH5α (ソ) | pSB1C3 | muta-CuMTS3 | ||
| 4 | DH5α (F) | pSB1C3 | muta-CuMTS3 | ||
| 5 | DH5α (N) | pSB1C3 | muta-CuMTS3 | ||
| 6 | 1kb Ladder | ||||
| 7 | DH5α (セ) | pSB1C3 | muta-CuMTS3 | EcoR1, Pst1 | |
| 8 | DH5α (ソ) | pSB1C3 | muta-CuMTS3 | EcoR1, Pst1 | |
| 9 | DH5α (F) | pSB1C3 | muta-CuMTS3 | EcoR1, Pst1 | |
| 10 | DH5α (N) | pSB1C3 | muta-CuMTS3 | EcoR1, Pst1 | |
| 11 | pSB1C3 | muta-CuMTS3 | EcoR1, Pst1 | ||
| 12 | 1kb Ladder | ||||
| 13 | pSB1C3 | Xba1 | |||
| 14 | DH5α (セ) | pSB1C3 | muta-CuMTS3 | Xba1 | |
| 15 | DH5α (ソ) | pSB1C3 | muta-CuMTS3 | Xba1 | |
| 16 | DH5α (F) | pSB1C3 | muta-CuMTS3 | Xba1 | |
| 17 | DH5α (N) | pSB1C3 | muta-CuMTS3 | Xba1 | |
Main Culture
| Sample | |||
|---|---|---|---|
| Host | Vector | Insert | Primer |
| DH5α(F) | pSB1C3 | muta-CuMTS3 | ---------- |
| DH5α(F) | pSB1C3 | muta-CuMTS3 | ---------- |
| DH5α(F) | pSB1C3 | muta-CuMTS3 | ---------- |
Cultivate these samples in 3mL of LB medium with ChlPhe at 30°C overnight.
September 21, 2014
KOBAYASHIMiniprep
| Sample | |||
|---|---|---|---|
| Host | Vector | Insert | Primer |
| DH5α(F) | pSB1C3 | muta-CuMTS3 | |
| DH5α(F) | pSB1C3 | muta-CuMTS3 | |
| DH5α(F) | pSB1C3 | muta-CuMTS3 | |
Restriction Digest
| Sample | Enzyme | Buffer | ||||
|---|---|---|---|---|---|---|
| Marker | Host | Vector | Insert | Primer | ||
| 1kb Ladder | ||||||
| pSB1C3 | BBa_J04450 | EcoR1 | Buffer H | |||
| pSB1C3 | BBa_J04450 | Pst1 | Buffer H | |||
| pSB1C3 | BBa_J04450 | EcoR1, Pst1 | Buffer H | |||
| pSB1C3 | BBa_J04450 | Spe1 | FD buffer | |||
| pSB1C3 | BBa_J04450 | Xba1 | FD buffer | |||
| pSB1C3 | BBa_J04450 | Spe1, Xba1 | FD buffer | |||
| pSB1C3 | BBa_J04450 | |||||
| λDNA-HindⅢ | ||||||
| F | pSB1C3 | muta-CuMTS3 | EcoR1 | Buffer H | ||
| F | pSB1C3 | muta-CuMTS3 | Pst1 | Buffer H | ||
| F | pSB1C3 | muta-CuMTS3 | EcoR1, Pst1 | Buffer H | ||
| F | pSB1C3 | muta-CuMTS3 | Spe1 | FD buffer | ||
| F | pSB1C3 | muta-CuMTS3 | Xba1 | FD buffer | ||
| F | pSB1C3 | muta-CuMTS3 | Spe1, Xba1 | FD buffer | ||
| F | pSB1C3 | muta-CuMTS3 | ||||
AGE
| Lane | Sample | Enzyme | ||||
|---|---|---|---|---|---|---|
| Marker | Host | Vector | Insert | Primer | ||
| 1 | 1kb Ladder | |||||
| 2 | pSB1C3 | BBa_J04450 | EcoR1 | |||
| 3 | pSB1C3 | BBa_J04450 | Pst1 | |||
| 4 | pSB1C3 | BBa_J04450 | EcoR1, Pst1 | |||
| 5 | pSB1C3 | BBa_J04450 | Spe1 | |||
| 6 | pSB1C3 | BBa_J04450 | Xba1 | |||
| 7 | pSB1C3 | BBa_J04450 | Spe1, Xba1 | |||
| 8 | pSB1C3 | BBa_J04450 | ||||
| 9 | λDNA-HindⅢ | |||||
| 10 | F | pSB1C3 | muta-CuMTS3 | EcoR1 | ||
| 11 | F | pSB1C3 | muta-CuMTS3 | Pst1 | ||
| 12 | F | pSB1C3 | muta-CuMTS3 | EcoR1, Pst1 | ||
| 13 | F | pSB1C3 | muta-CuMTS3 | Spe1 | ||
| 14 | F | pSB1C3 | muta-CuMTS3 | Xba1 | ||
| 15 | F | pSB1C3 | muta-CuMTS3 | Spe1, Xba1 | ||
| 16 | F | pSB1C3 | muta-CuMTS3 | |||
October
October 1, 2014
KOBAYASHIProtein Extraction (S. cerevisiae)
| Sample | |||
|---|---|---|---|
| Host | Vector | Insert | Primer |
| BY4247 | p427-TEF | ||
| BY4247 | p427-TEF | CuMTSE2 | pGEX6P-2F1, pGEX6P-2R |
| BY4247 | p427-TEF | CuMTS3 | pGEX6P-2F2, pGEX6P-2R |
| BY4247 | p427-TEF | CuMTS62 | pGEX6P-2F2, pGEX6P-2R |
SDS-PAGE
| Lane | Sample | |||
|---|---|---|---|---|
| Host | Vector | Insert | Primer | |
| 1 | BY4247 | p427-TEF | ||
| 2 | BY4247 | p427-TEF | CuMTSE2 | pGEX6P-2F1, pGEX6P-2R |
| 3 | BY4247 | p427-TEF | CuMTS3 | pGEX6P-2F2, pGEX6P-2R |
| 4 | BY4247 | p427-TEF | CuMTS62 | pGEX6P-2F2, pGEX6P-2R |
October 2, 2014
KOBAYASHIPreculture
| Sample | |||
|---|---|---|---|
| Host | Vector | Insert | Primer |
| BY4247 | p427-TEF | ||
| BY4247 | p427-TEF | CuMTS3 | pGEX6P-2F2, pGEX6P-2R |
Cultivate these samples in 3mL of SD medium with G418 at 30°C overnight.
Western blot
| Lane | Sample | |||
|---|---|---|---|---|
| Host | Vector | Insert | Primer | |
| 1 | BY4247 | p427-TEF | ||
| 2 | BY4247 | p427-TEF | CuMTSE2 | pGEX6P-2F1, pGEX6P-2R |
| 3 | BY4247 | p427-TEF | CuMTS3 | pGEX6P-2F2, pGEX6P-2R |
| 4 | BY4247 | p427-TEF | CuMTS62 | pGEX6P-2F2, pGEX6P-2R |
October 3, 2014
KOBAYASHIMain culture
Cultivate following samples in Big Scale with G418 at 28°C overnight in shaken culture.
| Sample | |||
|---|---|---|---|
| Host | Vector | Insert | Primer |
| BY4247 | p427-TEF | ||
| BY4247 | p427-TEF | CuMTS3 | pGEX6P-2F2, pGEX6P-2R |
Restriction Digest
| Sample | Enzyme | Buffer | ||
|---|---|---|---|---|
| Vector | Insert | Primer | ||
| pSB1C3 | EcoR1, Spe1 | Buffer H | ||
| pSB1C3 | Xba1, Pst1 | Buffer H | ||
| Linearized pSB1A3 | EcoR1, Pst1 | Buffer H | ||
AGE
| Lane | Marker | Sample | Enzyme | ||
|---|---|---|---|---|---|
| Vector | Insert | Primer | |||
| 1 | 1kb Ladder | ||||
| 2 | pSB1C3 | EcoR1, Spe1 | |||
| 3 | pSB1C3 | Xba1, Pst1 | |||
| 4 | Linearized pSB1A3 | EcoR1, Pst1 | |||
DNA Refinement 1
| Sample | Enzyme | ||
|---|---|---|---|
| Vector | Insert | Primer | |
| pSB1C3 | EcoR1, Spe1 | ||
| pSB1C3 | Xba1, Pst1 | ||
| pSB1C3 | Xba1, Pst1 | ||
| Linearized pSB1A3 | EcoR1, Pst1, Dpn1 | ||
Ligation
| Vector | Sample | Enzyme | |
|---|---|---|---|
| Insert1 | Insert2 | ||
| pSB1A3 | EcoR1, Xba1, Spe1, Pst1 | ||
| pSB1A3 | EcoR1, Xba1, Spe1, Pst1 | ||
DNA ligase: DNA Ligation Kit ‹Mighty Mix› (TAKARA BIO INC.)
Transformation
| Host | Sample | ||
|---|---|---|---|
| Vector | Insert1 | Insert2 | |
| DH5α | pSB1A3 | ||
| DH5α | pSB1A3 | ||
October 4, 2014
KOBAYASHIColony Isolation
Isolate each colony from the plates which were inoculated overnight.
| Host | Sample | Section | ||
|---|---|---|---|---|
| Vector | Insert | Primer | ||
| DH5α | pSB1A3 | 1-16 | ||
| DH5α | pSB1A3 | a-p | ||
Monoterpene Extraction
| Host | Sample | ||
|---|---|---|---|
| Vector | Insert | Primer | |
| BY4247 | p427-TEF | ||
| BY4247 | p427-TEF | CuMTS3 | pGEX6P-2F2, pGEX6P-2R |
Samples were stocked in -4℃.
Preculture
| Host | Sample | ||
|---|---|---|---|
| Vector | Insert | Primer | |
| BY4247 | p427-TEF | CuMTSE2 | pGEX6P-2F2, pGEX6P-2R |
| BY4247 | p427-TEF | CuMTS62 | pGEX6P-2F2, pGEX6P-2R |
Cultivate samples in 3mL of SD medium with G418 at 30°C overnight.
October 5, 2014
KOBAYASHIMiniprep
| Host | Sample | |
|---|---|---|
| Vector | Insert | |
| DH5α | pSB1A3 | |
| DH5α | pSB1A3 | |
Restriction Digest and AGE
| Host | Sample | Enzyme | |
|---|---|---|---|
| Vector | Insert | ||
| DH5α | pSB1C3 | EcoR1, Pst1 | |
| DH5α | pSB1C3 | EcoR1, Pst1 | |
| pSB1C3 | K1448000 | EcoR1, Pst1 | |
October 8, 2014
KOBAYASHIGC-MS analysis
| Host | Sample | ||
|---|---|---|---|
| Vector | Insert | Primer | |
| BY4247 | p427-TEF | ||
| BY4247 | p427-TEF | CuMTS3 | pGEX6P-2F2, pGEX6P-2R |
Preculture
| Host | Sample | ||
|---|---|---|---|
| Vector | Insert | Primer | |
| BY4247 | p427-TEF | ||
| BY4247 | p427-TEF | CuMTS3 | pGEX6P-2F2, pGEX6P-2R |
October 9, 2014
KOBAYASHIMain Culture
| Host | Sample | ||
|---|---|---|---|
| Vector | Insert | Primer | |
| BY4247 | p427-TEF | ||
| BY4247 | p427-TEF | CuMTS3 | pGEX6P-2F2, pGEX6P-2R |
October 10, 2014
KOBAYASHIGC Analysis
| Host | Sample | ||
|---|---|---|---|
| Vector | Insert | Primer | |
| BY4247 | p427-TEF | ||
| BY4247 | p427-TEF | CuMTS3 | pGEX6P-2F2, pGEX6P-2R |
