Difference between revisions of "Team:EPF Lausanne/Notebook/Ecoli"
| Line 149: | Line 149: | ||
<nav id="affix-nav" class="sidebar hidden-sm hidden-xs"> | <nav id="affix-nav" class="sidebar hidden-sm hidden-xs"> | ||
<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="active"><a href="# | + | <li class="active"><a href="#pdas9w">Make pdCas9-w</a> |
| + | <ul class="nav nav-pills nav-stacked" data-spy="affix" data-offset-top="200" data-offset-bottom="600"> | ||
| + | <li class="active"><a href="#PCRpdcas9">Open pdCas9 and extract w subunit from pWJ66 by PCR</a></li> | ||
| + | <li><a href="#gisbonpdcas9-w">Gibson assembly of pdCas9 and w subunit</a></li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li><a href="#pdcas9wsgrna">Make pdCas9-w-sgRNAs</a></li> | ||
</ul> | </ul> | ||
</nav> | </nav> | ||
| Line 156: | Line 162: | ||
<div class="col-sm-9"> | <div class="col-sm-9"> | ||
| − | <!-- --> | + | <!-- MAKE PDCAS9-W --> |
| − | <div id=" | + | <div id="pdcas9w" class="panel"> |
| − | + | <h1>Make pdCas9-w</h1> | |
| + | <p>Fuse dCas9 (from plasmid pdCas9) with the w subunit of polymerase (from plasmid pWJ66) to allow dCas9 to activate a gene when it binds upstream of the promoter.</p> | ||
| + | <p>We received the plasmids in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate the plasmids.</p> | ||
| + | <p>We opened pdCas9 and extracted the w subunit from pWJ66 by PCR. We assembled these parts with Gibson assembly.</p> | ||
</div> | </div> | ||
| + | |||
| + | <!-- PCR PDCAS9 --> | ||
| + | <div id="PCRpdcas9" class="panel"> | ||
| + | <h2>Open pdCas9 by PCR and extract w subunit from pWJ66 by PCR</h2> | ||
| + | <h3>05.06.2015</h3> | ||
| + | <h4>Materials and method</h4> | ||
| + | <p>Phusion PCR of 1 ng pdCas9 with HF buffer, annealing temperature (Ta): 58°C, extension time (ext): 150 sec, 30 cycles</p> | ||
| + | <p>Phusion PCR of 1 ng pWJ66 with HF buffer, annealing temperature (Ta): 62°C, extension time (ext): 15 sec, 30 cycles</p> | ||
| + | <p>PCR products were analyzed after PCR Product Purification (cf. Protocols) by 1,2% agarose gel electrophoresis.</p> | ||
| + | <h4>Results</h4> | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | </div> | ||
Revision as of 08:29, 24 July 2015
Protocols
Make pdCas9-w
Fuse dCas9 (from plasmid pdCas9) with the w subunit of polymerase (from plasmid pWJ66) to allow dCas9 to activate a gene when it binds upstream of the promoter.
We received the plasmids in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate the plasmids.
We opened pdCas9 and extracted the w subunit from pWJ66 by PCR. We assembled these parts with Gibson assembly.
Open pdCas9 by PCR and extract w subunit from pWJ66 by PCR
05.06.2015
Materials and method
Phusion PCR of 1 ng pdCas9 with HF buffer, annealing temperature (Ta): 58°C, extension time (ext): 150 sec, 30 cycles
Phusion PCR of 1 ng pWJ66 with HF buffer, annealing temperature (Ta): 62°C, extension time (ext): 15 sec, 30 cycles
PCR products were analyzed after PCR Product Purification (cf. Protocols) by 1,2% agarose gel electrophoresis.