Difference between revisions of "Team:EPF Lausanne/Notebook/Ecoli"

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                         <h2>Results</h2>
 
                         <h2>Results</h2>
 
                             <img src="https://static.igem.org/mediawiki/2015/6/6e/10.05_pcr_pdcas9.jpg" align="center">
 
                             <img src="https://static.igem.org/mediawiki/2015/6/6e/10.05_pcr_pdcas9.jpg" align="center">
                             <p><small>After testing many parameters, we were able to amplify the plasmid succesfully, as seen on gel above.</br>For further steps sample from lane 1 was used. It was prepared with HF buffer and the following thermocycling settings:</small></p>
+
                             <p><small>After testing many parameters, we were able to amplify the plasmid succesfully, as seen on gel above (Date: 05/10/15).</br>For further steps 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>
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                                     <td>  </td>
 
                                     <td>  </td>
 
                                     <td>98°C</td>
 
                                     <td>98°C</td>
                                     <td><mark>40 seconds</mark></td>
+
                                     <td>40 seconds</td>
 
                                 </tr>
 
                                 </tr>
 
                                 <tr>
 
                                 <tr>
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                                     <td>Denaturation</td>
 
                                     <td>Denaturation</td>
 
                                     <td>98°C</td>
 
                                     <td>98°C</td>
                                     <td><mark>15 seconds</mark></td>
+
                                     <td>15 seconds</td>
 
                                 </tr>
 
                                 </tr>
 
                                 <tr>
 
                                 <tr>
 
                                     <td>  </td>
 
                                     <td>  </td>
 
                                     <td>Annealing</td>
 
                                     <td>Annealing</td>
                                     <td><mark>59°C</mark></td>
+
                                     <td>59°C</td>
                                     <td><mark>22 seconds</mark></td>
+
                                     <td>22 seconds</td>
 
                                 </tr>
 
                                 </tr>
 
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                                     <td>Extension</td>
 
                                     <td>Extension</td>
 
                                     <td>72°C</td>
 
                                     <td>72°C</td>
                                     <td><mark>2 minutes 30 seconds</mark></td>
+
                                     <td>2 minutes 30 seconds</td>
 
                                 </tr>
 
                                 </tr>
 
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                                     <td>  </td>
 
                                     <td>  </td>
 
                                     <td>72°C</td>
 
                                     <td>72°C</td>
                                     <td><mark><small>7 minutes</small></mark></td>
+
                                     <td>7 minutes</td>
 
                                 </tr>
 
                                 </tr>
 
                                 <tr>
 
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Revision as of 13:58, 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

After testing many parameters, we were able to amplify the plasmid succesfully, as seen on gel above (Date: 05/10/15).
For further steps 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 will extract the w subunit to fuse it to our own dCas9.

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

Still under construction