Difference between revisions of "Team:EPF Lausanne/Notebook/Ecoli"
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<ul class="nav nav-pills nav-stacked" data-spy="affix" data-offset-top="200" data-offset-bottom="600"> | <ul class="nav nav-pills nav-stacked" data-spy="affix" data-offset-top="200" data-offset-bottom="600"> | ||
<li class="dropdown"> | <li class="dropdown"> | ||
| − | <a class="dropdown-toggle" data-toggle="dropdown" href="#"> | + | <a class="dropdown-toggle" data-toggle="dropdown" href="#">Assemble pdCas9-w<span class="caret"></span></a> |
<ul class="dropdown-menu"> | <ul class="dropdown-menu"> | ||
| − | <li><a href="#PCRpdcas9">PCR</a></li> | + | <li><a href="#PCRpdcas9">Open pdCas9 by PCR</a></li> |
| − | <li><a href="# | + | <li><a href="#PCRpwj66">Extract w subunit from pWJ66 by PCR</a></li> |
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| − | <!-- PCR PDCAS9 | + | <!-- PCR PDCAS9--> |
<div id="PCRpdcas9" class="panel"> | <div id="PCRpdcas9" class="panel"> | ||
| − | <h1> | + | <h1>Assemble pdCas9-w:</br>Open pdCas9 by PCR</h2> |
| − | <p><small>We received | + | <p><small>We received plasmid pdCas9 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate it.</small></p> |
| − | <h2 | + | <h2>Materials and method</h2> |
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<ul> | <ul> | ||
| − | <li>20 µl Phusion PCR (cf. Protocols) with 1 | + | <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> |
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<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> | ||
</ul> | </ul> | ||
| − | + | <h2>Results</h2> | |
| − | + | <img src="https://static.igem.org/mediawiki/2015/6/6e/10.05_pcr_pdcas9.jpg" align="middle"> | |
| − | + | <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> | |
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| + | <!-- PCR PWJ66--> | ||
| + | <div id="PCRpwj66" class="panel"> | ||
| + | <h1>Assemble pdCas9-w:</br>Extract w subunit from pWJ66 by PCR</h2> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/e/ee/7.05_pcr_gel.jpg" align="middle"> | ||
</div> | </div> | ||
Revision as of 14:55, 30 July 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.
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.
