Difference between revisions of "Team:EPF Lausanne/Notebook/Yeast"
| Line 38: | Line 38: | ||
<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="#integrate_pTPGI_dCas9_VP64">Integrate pTPGI_dCas9_VP64</a></li> | <li class="active"><a href="#integrate_pTPGI_dCas9_VP64">Integrate pTPGI_dCas9_VP64</a></li> | ||
| − | < | + | </u1> |
| + | |||
<li <a href="#Integrate_reporter_plasmid">Integrate reporter plasmid</a></li> | <li <a href="#Integrate_reporter_plasmid">Integrate reporter plasmid</a></li> | ||
</u1> | </u1> | ||
| Line 46: | Line 47: | ||
<li><a href="#integrate_reporters_plasmids">Integrate the reporter plasmids</a></li> | <li><a href="#integrate_reporters_plasmids">Integrate the reporter plasmids</a></li> | ||
<ul> | <ul> | ||
| + | |||
<li><a href="#Integrate_and_express_gRNAs">Integrate and express gRNAs</a></li> | <li><a href="#Integrate_and_express_gRNAs">Integrate and express gRNAs</a></li> | ||
</ul> | </ul> | ||
| Line 73: | Line 75: | ||
<div id="integrate_pTPGI_dCas9_VP64" class="panel"> | <div id="integrate_pTPGI_dCas9_VP64" class="panel"> | ||
<h1><small>Integrate pTPGI_dCas9_VP64</small></br>Integrate pTPGI_dCas9_VP64</h1> | <h1><small>Integrate pTPGI_dCas9_VP64</small></br>Integrate pTPGI_dCas9_VP64</h1> | ||
| − | <p><small>We received plasmid pTPGI_dCas9_VP64 in bacterial stabs. This step consists in | + | <p><small>We received plasmid pTPGI_dCas9_VP64 in bacterial stabs. This step consists in linearising pTPGI_dCas9_VP64 by PCR.</small></p> |
<h2>Materials and method</h2> | <h2>Materials and method</h2> | ||
Revision as of 16:18, 17 August 2015
saccharomyces cerevisiae
pTPGI_dCas9_VP64 integration
As we need dCas9 expressing yeasts, we decided to integrate a plasmid that would inducively express dCas9.
First and second trial
The negative and positive control worked fine, but the transformation control didn’t work. After investigation we found out that we didn’t have an origin of replication in our plasmid thus not enabling our yeasts to replicate the plasmid and to grow. See more details here
Integrate pTPGI_dCas9_VP64Integrate pTPGI_dCas9_VP64
We received plasmid pTPGI_dCas9_VP64 in bacterial stabs. This step consists in linearising pTPGI_dCas9_VP64 by PCR.
Materials and method
- Glycerol stocks
- Miniprep
- Restriction analysis
- Integration into yeast genome
Results
Restriction analysis confirms we have the right plasmid.<\br> Linearized pdCas9-w is expected to be 6705 bp.<\br> The first try of this PCR was unsuccessful (gel not shown here). For our second try, we tested many parameters: HF vs. GC buffer, different annealing temperatures and different extension times. This time, many, but not all, of our samples were successfully amplified (cf. figure 1). The difficulty of this PCR is probably due to the fact that the size of the ampicon is very long, almost 7 kb.For next steps, sample from lane 1 (cf. figure 1) was used.
