Difference between revisions of "Team:EPF Lausanne/Notebook/Ecoli"
| Line 173: | Line 173: | ||
</ul> | </ul> | ||
<h2>Results</h2> | <h2>Results</h2> | ||
| − | <img src="https://static.igem.org/mediawiki/2015/6/6e/ | + | <img src="https://static.igem.org/mediawiki/2015/archive/6/6e/20150804142724%2110.05_pcr_pdcas9.jpg" align="center"> |
| − | <p><small>After testing many parameters, we were able to | + | <p><small>Linearized pdCas9-w is expected to be 6705 bp.</br>After testing many parameters, we were able to linearize the plasmid succesfully, as seen on gel above.</br>For further uses, sample from lane 1 was used. It was prepared with HF buffer and the following thermocycling settings:</small></p> |
<table width="70%" align="center"> | <table width="70%" align="center"> | ||
<tr> | <tr> | ||
| Line 224: | Line 224: | ||
<div id="PCRpwj66" class="panel"> | <div id="PCRpwj66" class="panel"> | ||
<h1>Assemble pdCas9-w: Extract w subunit from pWJ66 by PCR</h1> | <h1>Assemble pdCas9-w: Extract w subunit from pWJ66 by PCR</h1> | ||
| − | <p><small>We received plasmid pWJ66 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate it.</br>pWJ66 is a plasmid containing dCas9 fused at its C-terminal to the w subunit of RNA polymerase as well as a tracrRNA gene and a CRISPR array gene, available on Addgene. We | + | <p><small>We received plasmid pWJ66 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate it.</br>pWJ66 is a plasmid containing dCas9 fused at its C-terminal to the w subunit of RNA polymerase as well as a tracrRNA gene and a CRISPR array gene, available on Addgene. We extracted the w subunit to fuse it to our own dCas9.</small></p> |
<h2>Materials and method</h2> | <h2>Materials and method</h2> | ||
<ul> | <ul> | ||
| − | <li>20 µl Phusion PCR (cf. Protocols) with 1 ng | + | <li>20 µl Phusion PCR (cf. Protocols) with 1 ng pWJ66 using HF buffer and following thermocycling settings:</li> |
| + | <table width="70%" align="center"> | ||
| + | <tr> | ||
| + | <th>Step</th> | ||
| + | <th> </th> | ||
| + | <th>Temperature</th> | ||
| + | <th>Time</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Initial Denaturation</td> | ||
| + | <td> </td> | ||
| + | <td>98°C</td> | ||
| + | <td>30 seconds</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>30 cycles</td> | ||
| + | <td>Denaturation</td> | ||
| + | <td>98°C</td> | ||
| + | <td>10 seconds</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> </td> | ||
| + | <td>Annealing</td> | ||
| + | <td>62°C</td> | ||
| + | <td>15 seconds</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> </td> | ||
| + | <td>Extension</td> | ||
| + | <td>72°C</td> | ||
| + | <td>15 seconds</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Final Extension</td> | ||
| + | <td> </td> | ||
| + | <td>72°C</td> | ||
| + | <td>7 minutes</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Hold</td> | ||
| + | <td> </td> | ||
| + | <td>4°C </td> | ||
| + | <td> </td> | ||
| + | </tr> | ||
| + | </table> | ||
<li>PCR product purification (cf. Protocols)</li> | <li>PCR product purification (cf. Protocols)</li> | ||
<li>Agarose gel electrophoresis of purified PCR products</li> | <li>Agarose gel electrophoresis of purified PCR products</li> | ||
Revision as of 14:36, 4 August 2015
E. Coli Laboratory Notebook
Assemble pdCas9-w: Open pdCas9 by PCR
We received plasmid pdCas9 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate it.pdCas9 is a plasmid containing dCas9 under a Tetracyclin inducible promoter and a Chloramphenicol resistance gene, available on Addgene under the name pdCas9-bacteria.
Materials and method
- 20 µl Phusion PCR (cf. Protocols) with 1 ng pdCas9 testing parameters such as HF vs. GC buffer, annealing temperatures and extension times
- PCR product purification (cf. Protocols)
- Agarose gel electrophoresis of purified PCR products
Results
Linearized pdCas9-w is expected to be 6705 bp.After testing many parameters, we were able to linearize the plasmid succesfully, as seen on gel above.For further uses, sample from lane 1 was used. It was prepared with HF buffer and the following thermocycling settings:
| Step | Temperature | Time | |
|---|---|---|---|
| Initial Denaturation | 98°C | 40 seconds | |
| 25 cycles | Denaturation | 98°C | 15 seconds |
| Annealing | 59°C | 22 seconds | |
| Extension | 72°C | 2 minutes 30 seconds | |
| Final Extension | 72°C | 7 minutes | |
| Hold | 4°C |
Assemble pdCas9-w: Extract w subunit from pWJ66 by PCR
We received plasmid pWJ66 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate it.pWJ66 is a plasmid containing dCas9 fused at its C-terminal to the w subunit of RNA polymerase as well as a tracrRNA gene and a CRISPR array gene, available on Addgene. We extracted the w subunit to fuse it to our own dCas9.
Materials and method
- 20 µl Phusion PCR (cf. Protocols) with 1 ng pWJ66 using HF buffer and following thermocycling settings:
- PCR product purification (cf. Protocols)
- Agarose gel electrophoresis of purified PCR products
| Step | Temperature | Time | |
|---|---|---|---|
| Initial Denaturation | 98°C | 30 seconds | |
| 30 cycles | Denaturation | 98°C | 10 seconds |
| Annealing | 62°C | 15 seconds | |
| Extension | 72°C | 15 seconds | |
| Final Extension | 72°C | 7 minutes | |
| Hold | 4°C |
Results
