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

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            <!-- Experience 1 -->
 
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                    <h3>pTPGI_dCas9_VP64 integration</h3>
 
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                        <p>As we need dCas9 expressing yeasts, we decided to integrate a plasmid that would inducively express dCas9.</p>
 
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                        <h5>First and second trial</h5>
 
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                            <p>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.
 
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                            See more details <a href="https://static.igem.org/mediawiki/2015/0/06/EPF_Lausanne_trial1pTPGI_dCas9_VP64integration.pdf">here</a></p>
 
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     <!--PCR PDCAS9-->
 
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        <h1><small>Integrate pTPGI_dCas9_VP64</small></br>Integrate pTPGI_dCas9_VP64</h1>
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    <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 bacterial stabs. This step consists in linearising pTPGI_dCas9_VP64 by PCR.</small></p>
  
 
             <h2>Materials and method</h2>
 
             <h2>Materials and method</h2>
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             <h2>Results</h2>
 
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Revision as of 16:41, 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 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.

EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits