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">
 
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                         <li class="active"><a href="#pdas9w">Make pdCas9-w</a>
 
                         <li class="active"><a href="#pdas9w">Make pdCas9-w</a>
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                                 <li class="active"><a href="#PCRpdcas9">Open pdCas9 and extract w subunit from pWJ66 by PCR</a></li>
 
                                 <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>
 
                                 <li><a href="#gisbonpdcas9-w">Gibson assembly of pdCas9 and w subunit</a></li>

Revision as of 13:21, 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.

Results

Still under construction