Team:Warsaw/Calendar-Main/24 September 2008

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(New page: {{WarNotebook}} <!-- do not edit above me! --> <html> <h3>Protein A mutagenesis<br>Paweł</h3> <p><ol><li>Results of mutagenesis: no colonies on each plate. </li> <li>Mutagenesis repeat...)
 
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<h3>Protein A mutagenesis<br>Paweł</h3>
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<h3>MutD<sub>5</sub> testing</h3>
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<h4>Emilia</h4>
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<ol><li>
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<p>Inoculation MutD<sub>5</sub> from previous day to new medium (liquid LB + 0.25mM IPTG + kanamycin + tetracyclin + 50 μl/ml + prey)
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</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>
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</p>
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<p><ol><li>Results of mutagenesis: no colonies on each plate. </li>
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</ol>
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<h3>Mutagenesis of protein A</h3><h4>Paweł</h4>
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<p><ol><li>Results of mutagenesis: no colonies on any plate. </li>
<li>Mutagenesis repeated with modified conditions: fresh stock of dNTPs used and additional MgCl2 added.</li></ol></p>
<li>Mutagenesis repeated with modified conditions: fresh stock of dNTPs used and additional MgCl2 added.</li></ol></p>
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<h3>Preparation of alpha_A construct</h3>
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<h4>Antoni</h4>
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<p>
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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> on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII+A>pKS plasmid containing protein A</a> with
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+link10+homo2">AL+link10+homo2</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a>
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primers and 10% DMSO(20 cycles, elongation 40&nbsp;s, annealing temperature 72&deg;C). </li>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=http://www.fermentas.com/techinfo/nucleicacids/mappuc1819.htm>pUC19</a> plasmid with
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaL+SacI">AlphaL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaP+link10+homo2">AlphaP+link10+homo2</a>
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primers (20 cycles, elongation 45&nbsp;s, annealing temperature 63&deg;C).
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<br>
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As a result we got two PCR products (alpha-linker and linker-A) wich will be utilized as templates in next PCR. </li>
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<li> Gel electrophoresis of PCR products and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands (alpha_linker - 570 bp and linker_A - 470 bp ).</li>
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<li>Measurment of concentration of both isolated products.</li>
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</ol></p>
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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>
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<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 <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>
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<li>Plating on LB + ampicillin.</li>
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</ol>

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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