Difference between revisions of "Team:EPF Lausanne/Notebook/Yeast"

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     <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>
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         <p><small>We received plasmid pTPGI_dCas9_VP64 in bacterial stabs. This step consists in linearising pTPGI_dCas9_VP64 by PCR.</small></p>
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         <p><small>We received plasmid pTPGI_dCas9_VP64 in bacteria from Addgene. We inoculated single colonies of bacteria in order to prepare glycerol stocks and minipreps. We performed a restriction analysis to check the identity of our plasmid. We linearised the plasmid by PCR, in order to integrate it into yeast genome.</small></p>
  
 
             <h2>Materials and method</h2>
 
             <h2>Materials and method</h2>
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                     <li>Miniprep</li>
 
                     <li>Miniprep</li>
 
                     <li>Restriction analysis</li>
 
                     <li>Restriction analysis</li>
 +
                    <li>Polymerase Chain Reaction</li>
 
                     <li>Integration into yeast genome</li>
 
                     <li>Integration into yeast genome</li>
 
                 </ul>
 
                 </ul>
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                 </div>
 
                 </div>
 
                 <div id="divright1">
 
                 <div id="divright1">
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                     <p><small>Restriction analysis confirms we have the right plasmid.</br>
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                     <p><small>We used four different sets of enzymes for the restriction analysis. Linearized pTPGI_dCas9_VP64 is expected to be 10'987 bp. We observe that the gel corresponds to the expected one (fig. 2). </br>
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                              Linearized pdCas9-w is expected to be 6705 bp.</br>
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                               The plasmid was linearised both with EagI HF and NotI HF prior to integration. We integrated each linearised plasmid to obtain two different yeast strains.
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                               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.</br>For next steps, sample from lane 1 (cf. figure 1) was used.</small></p>
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                              </small></p>
 
                 </div>
 
                 </div>
 
     </div>
 
     </div>

Revision as of 17:09, 17 August 2015

EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits

saccharomyces cerevisiae

Integrate pTPGI_dCas9_VP64
Integrate pTPGI_dCas9_VP64

We received plasmid pTPGI_dCas9_VP64 in bacteria from Addgene. We inoculated single colonies of bacteria in order to prepare glycerol stocks and minipreps. We performed a restriction analysis to check the identity of our plasmid. We linearised the plasmid by PCR, in order to integrate it into yeast genome.

Materials and method

  • Glycerol stocks
  • Miniprep
  • Restriction analysis
  • Polymerase Chain Reaction
  • Integration into yeast genome

Results

We used four different sets of enzymes for the restriction analysis. Linearized pTPGI_dCas9_VP64 is expected to be 10'987 bp. We observe that the gel corresponds to the expected one (fig. 2).
The plasmid was linearised both with EagI HF and NotI HF prior to integration. We integrated each linearised plasmid to obtain two different yeast strains.

EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits