Difference between revisions of "Team:EPF Lausanne/Notebook/Ecoli"
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<!-- PCR PDCAS9--> | <!-- PCR PDCAS9--> | ||
<div id="PCRpdcas9" class="panel"> | <div id="PCRpdcas9" class="panel"> | ||
| − | <h1>Assemble pdCas9-w: | + | <h1>Assemble pdCas9-w: Open pdCas9 by PCR</h2> |
| − | <p><small>We received plasmid pdCas9 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate it.</small></p> | + | <p><small>We received plasmid pdCas9 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate it.</br>pdCas9 is a plasmid containing dCas9 under a Tetracyclin inducible promoter and a Chloramphenicol resistance gene, available on Addgene under the name pdCas9-bacteria.</small></p> |
<h2>Materials and method</h2> | <h2>Materials and method</h2> | ||
<ul> | <ul> | ||
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</ul> | </ul> | ||
<h2>Results</h2> | <h2>Results</h2> | ||
| − | <img src="https://static.igem.org/mediawiki/2015/6/6e/10.05_pcr_pdcas9.jpg | + | <img src="https://static.igem.org/mediawiki/2015/6/6e/10.05_pcr_pdcas9.jpg"> |
<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. Its parameters were: HF buffer, annealing temperature 59°C, extension time 2 minutes 30 seconds.</small></p> | <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. Its parameters were: HF buffer, annealing temperature 59°C, extension time 2 minutes 30 seconds.</small></p> | ||
</div> | </div> | ||
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<!-- PCR PWJ66--> | <!-- PCR PWJ66--> | ||
<div id="PCRpwj66" class="panel"> | <div id="PCRpwj66" class="panel"> | ||
| − | <h1>Assemble pdCas9-w: | + | <h1>Assemble pdCas9-w: Extract w subunit from pWJ66 by PCR</h1> |
| − | <img src="https://static.igem.org/mediawiki/2015/e/ee/7.05_pcr_gel.jpg" | + | <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 will extract the w subunit to fuse it to our own dCas9.</small></p> |
| + | <h2>Materials and method</h2> | ||
| + | <ul> | ||
| + | <li>20 µl Phusion PCR (cf. Protocols) with 1 ng pdCas9 testing parameters such as HF vs. GC buffer, annealing temperatures and extension times</li> | ||
| + | <li>PCR product purification (cf. Protocols)</li> | ||
| + | <li>Agarose gel electrophoresis of purified PCR products</li> | ||
| + | </ul> | ||
| + | <h2>Results</h2> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/e/ee/7.05_pcr_gel.jpg"> | ||
| + | <p> | ||
</div> | </div> | ||
Revision as of 12:25, 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.For further steps sample from lane 1 was used. Its parameters were: HF buffer, annealing temperature 59°C, extension time 2 minutes 30 seconds.
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
