Team:Warsaw/Calendar-Main/24 September 2008

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<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 constructs with alpha-A</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 pKS plasmid containing protein 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 (20 cycles, elongation duration 15 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_XB">AlphaP_XB</a>
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primers (20 cycles, elongation duration 45 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 - 572 bp and linker_A - 467 bp ).</li>
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<li>Measurment of concentration of both isolated products</li>
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</ol></p>
</html>
</html>

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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 constructs with alpha-A

Antoni

  1. PCR on pKS plasmid containing protein A with AL+link10+homo2 and AP+NotI primers (20 cycles, elongation duration 15 s, annealing temperature 72°C).
  2. PCR on pUC19 plasmid with AlphaL+SacI and AlphaP_XB primers (20 cycles, elongation duration 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 - 572 bp and linker_A - 467 bp ).
  4. Measurment of concentration of both isolated products

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