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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<p>Reason of white phenotype unknown.</p>
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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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<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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<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>
<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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<h3>PCR results of translational fusion for the pMPMT5-AID+AIDT7 construct.<br> Lane 1 - 25 cycles, lane 2 - 20 cycles.</h3>
 
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<img src="https://static.igem.org/mediawiki/2008/0/08/Tranlacyjna_pcr_29_05_2008.jpg">
 

Revision as of 16:13, 8 October 2008

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Sequencing determined that all white colonies have intact lacZ gene.

Reason of white phenotype unknown.

We suppose that it is due to chromosomal mutation.

We need another reporter system (not splitted to chromosomal and plasmid part --> GFP? ).

PCR results

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

Preparation of pMPMT5-AID+AIDT7 construct

  1. PCR - translation fusion: AID + T7 RNA-polimerase
    Primers: AIDlNrH and T7pXbSal
    Template DNA: AID for translation fusion and T7 RNA-polimerase for translation fusion
    Elongation temperature: 55°C
    Elongation time: 4 minutes
    Number of cycles: 20 and 25 (2 repeats)
  2. Gel electrophoresis and DNA isolation from proper band (obtained with 20 cycles)

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