Team:Warsaw/Calendar-Main/23 September 2008

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<h3>Protein A mutagenesis<br>Paweł</h3>
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<h3>Mutagenesis of protein A</h3><h4>Paweł</h4>
<p><ol><li>PNK phosporylation of mutageneses for 30 minutes at 37C.</li>
<p><ol><li>PNK phosporylation of mutageneses for 30 minutes at 37C.</li>
<li>PNK heat-inactivated by incubation at 75C for 15 minutes.</li>
<li>PNK heat-inactivated by incubation at 75C for 15 minutes.</li>
<li>T4 ligase added and incubated for 1h at room temperature.</li>
<li>T4 ligase added and incubated for 1h at room temperature.</li>
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<li>T4 ligase inactivated by incubation at 55C for 20 min, then 5U of DpnI added and incubated at 37C for 3 hours.</li> <li>After DpnI treatment mutageneses transformed into TOP10 and plated on LB+kan plates.</li></ol></p>
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<li>T4 ligase inactivated by incubation at 55C for 20 min, then 5U of DpnI added and incubated at 37C for 3 hours.</li> <li>After DpnI treatment mutageneses transformed into TOP10 and plated on LB + kanamycin plates.</li></ol></p>
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Mutagenesis of protein A

Paweł

  1. PNK phosporylation of mutageneses for 30 minutes at 37C.
  2. PNK heat-inactivated by incubation at 75C for 15 minutes.
  3. T4 ligase added and incubated for 1h at room temperature.
  4. T4 ligase inactivated by incubation at 55C for 20 min, then 5U of DpnI added and incubated at 37C for 3 hours.
  5. After DpnI treatment mutageneses transformed into TOP10 and plated on LB + kanamycin plates.

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