Team:Warsaw/Calendar-Main/24 September 2008

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<h3>MutD<sub>5</sub> testing</h3>
<h3>MutD<sub>5</sub> testing</h3>
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<h4>Piotr</h4>
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<h4>Emilia</h4>
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<p>Inoculation MutD<sub>5</sub> from previous day to the same medium (liquid LB + 0.25mM IPTG + kanamycin + 50 μl/ml + prey)
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<ol><li>
 +
<p>Inoculation MutD<sub>5</sub> from previous day to new medium (liquid LB + 0.25mM IPTG + kanamycin + tetracyclin + 50 μl/ml + prey)
 +
</li><li>6 hours culture</li><li>
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Inoculation MutD<sub>5</sub> from the morning to new medium (liquid LB + 0.25mM IPTG + kanamycin + tetracyclin + 50 μl/ml + prey)</li>
</p>
</p>
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</ol>
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</ol></p>
</ol></p>
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<h3>Preparation of BioBricks</h3>
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<h3>Preparation of <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>&Delta;A (BBa_K103003)</a></h3>
<h4>Piotr</h4>
<h4>Piotr</h4>
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<ol><li> <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">transformation</a> with overnight ligation of RFP(??????) and deltaA DNA fragments. </li>
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<ol><li> <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">transformation</a> with overnight ligation of <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> and <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>&Delta;A</a> DNA fragments. </li>
<li>Plating on LB + ampicillin.</li>
<li>Plating on LB + ampicillin.</li>

Latest revision as of 03:46, 29 October 2008

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MutD5 testing

Emilia

  1. Inoculation MutD5 from previous day to new medium (liquid LB + 0.25mM IPTG + kanamycin + tetracyclin + 50 μl/ml + prey)

  2. 6 hours culture
  3. Inoculation MutD5 from the morning to new medium (liquid LB + 0.25mM IPTG + kanamycin + tetracyclin + 50 μl/ml + prey)

Mutagenesis of protein A

Paweł

  1. Results of mutagenesis: no colonies on any plate.
  2. Mutagenesis repeated with modified conditions: fresh stock of dNTPs used and additional MgCl2 added.

Preparation of alpha_A construct

Antoni

  1. PCR on pKS plasmid containing protein A with AL+link10+homo2 and AP+NotI primers and 10% DMSO(20 cycles, elongation 40 s, annealing temperature 72°C).
  2. PCR on pUC19 plasmid with AlphaL+SacI and AlphaP+link10+homo2 primers (20 cycles, elongation 45 s, annealing temperature 63°C).
    As a result we got two PCR products (alpha-linker and linker-A) wich will be utilized as templates in next PCR.
  3. Gel electrophoresis of PCR products and gel-out of proper bands (alpha_linker - 570 bp and linker_A - 470 bp ).
  4. Measurment of concentration of both isolated products.

Preparation of ΔA (BBa_K103003)

Piotr

  1. E. coli TOP10 transformation with overnight ligation of pSB1A3 and ΔA DNA fragments.
  2. Plating on LB + ampicillin.

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