Team:Warsaw/Calendar-Main/3 September 2008

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<li>A-alpha, Z-alpha and Z-omega inoculated in the overnight culture (Rosetta strain)</li></ol></p>
<li>A-alpha, Z-alpha and Z-omega inoculated in the overnight culture (Rosetta strain)</li></ol></p>
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<h3>Protein A mutagenesis<br>Paweł</h3>
 
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<p><ol><li>PNK phosporylation of mutageneses for 30 minutes at 37C.</li>
 
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<li>PNK heat-inactivated by incubation at 75C for 15 minutes.</li>
 
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<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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Purification of proteins: A-alpha, Z-alpha and Z-omega
Piotr

  1. Samples were resuspended in PBS, sonicated and centrifuged.
  2. Lysis buffer were added separately to pellet and supernatant, then samples were boiled for 10 minutes.
  3. Lysates loaded on 12% poliacrylamide gel (amount relating to 100 ul of OD=1,0 culture)
  4. Gel stained with Coomassie Blue. Optimal induction conditions chosen
  5. A-alpha, Z-alpha and Z-omega inoculated in the overnight culture (Rosetta strain)

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