Team:Warsaw/Calendar-Main/24 June 2008

From 2008.igem.org

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<p>Sequencing determined that all white colonies have intact lacZ gene.</p>
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<h3>Preparation of alpha-A and omega-A fusions</h3>
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<p>Reason of white phenotype unknown.</p>
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<h4>Michał L., Ewa, Marcin</h4>
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<p>We suppose that it is due to chromosomal mutation.</p>
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<p>We need another reporter system (not splitted to chromosomal and plasmid part --> GFP? ).</p>
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<p>Still no success. We need to run gradient PCR to find optimal reaction conditions.</p>
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<h3>PCR results</h3>
 
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Still no success. We need to run gradient PCR to find optimal reaction conditions.
 
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<h3>Preparation of constructs: OmpA_alpha and OmpA_omega</h3>
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<h4>Michał K.</h4>
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<ol><li> PCR on isolated plasmids with <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaL_N">OmpaL_N</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaP_link">OmpaP_link</a>
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primers.</li>
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<li>Gel electrophoresis (<a href="https://2008.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/24_June_2008#fig1">Fig. 1</a>, no proper clones found).</li>
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</ol>
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<h3>Preparation of pMPMT5-AID+AIDT7 construct</h3>
 
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<ol>
 
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<li><a href=<"https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr>PCR</a> - translation fusion: AID + T7 RNA-polymerase <br>
 
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Primers:
 
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDlNrH">AIDlNrH</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7pXbSal">T7pXbSal</a><br>
 
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Template DNA: AID for translation fusion and T7 RNA-polymerase for translation fusion<br>
 
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Elongation temperature: 55&deg;C<br>
 
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Elongation time: 4 minutes<br>
 
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Number of cycles: 20 and 25 (2 repeats)</li>
 
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<li>Gel electrophoresis and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">DNA isolation</a> from proper band (obtained with 20 cycles)</li>
 
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<br>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/7/78/Omp_colony_PCR_WAW.jpg" width=350/></a>
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<var><b>Fig. 1.</b> PCR product - searching for fusions with OmpA protein. Lower lanes - OmpA_alpha, upper - OmpA_omega.</var>

Latest revision as of 16:53, 28 October 2008

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Preparation of alpha-A and omega-A fusions

Michał L., Ewa, Marcin

Still no success. We need to run gradient PCR to find optimal reaction conditions.

Preparation of constructs: OmpA_alpha and OmpA_omega

Michał K.

  1. PCR on isolated plasmids with OmpaL_N and OmpaP_link primers.
  2. Gel electrophoresis (Fig. 1, no proper clones found).

Fig. 1. PCR product - searching for fusions with OmpA protein. Lower lanes - OmpA_alpha, upper - OmpA_omega.

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