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="active"><a href="#pdas9w">Make pdCas9-w</a> | <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> | ||
<li><a href="#pdcas9wsgrna">Make pdCas9-w-sgRNAs</a></li> | <li><a href="#pdcas9wsgrna">Make pdCas9-w-sgRNAs</a></li> | ||
Revision as of 13:24, 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.