Team:Warsaw/Calendar-Main/2 September 2008

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<h3>Purification of proteins: A-alpha, Z-alpha and Z-omega<br>
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<h3>Purification of proteins: A-alpha</h3><h4>Piotr, Emilia</h4>
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Piotr </h3>
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<p><ol><li><a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BA-alpha>pET15b+A_alpha</a> in <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosetta</a> strain from overnight culture inoculated in fresh LB and cultured until OD=0.5.</li>
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<p><ol><li>A-alpha, Z-alpha and Z-omega from overnight culture inoculated in fresh LB and cultured until OD=0,5</li>
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<li>Culture was induced with different concentrations of IPTG at 22&deg;C and 37&deg;C.</li>
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<li>Cultures induced with different concentrations of IPTG in 22C and 37C.</li>
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<li>Samples were collected twice: after 3h and next day (samples were centrifuged and frozen).</li></ol></p>
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<li>Samples were collected twice: after 3h and next day (samples were centrifuged and frozen)</li></ol></p>
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<h3>Mutagenesis of protein A<br>Paweł</h3>
 
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<p>Mutagenesis of protein A was performed using 2 pairs of primers: <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#ADelL+KpnI>ADelL+KpnI</a> and <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#ADelP>ADelP</a> (deletion of aminoacids involved in interaction with protein Z) and <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#AmutL+KpnI>AMutL+KpnI</a>  and <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#AmutP>AMutP</a> (changing of few aminoacids involved in interaction with protein Z).</p>
 
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<p>Mutagenesis were performed on 3 vectors:  pACYClac+ompA-A-omega, pACYClac+ompa-A-alpha and pACYClac+ ompa-alpha-A.</p>
 
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<p>Each mutagenesis were performed using each primer at final concentration 0,1 uM, dNTPs at final concentration 0,25 uM and Walk (Pwo) polymerase (shipped by <a href=http://aabiot.com/>A&A Biotechnology</a>), with 100 ng of DNA template. </p>
 
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<p><br>PCR program (15 cycles):
 
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<pre style="text-align: left">94C 5 min
 
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94C 30 s
 
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55C 30 s
 
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72C 10 min
 
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72C 8 min
 
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4C pause
 
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</pre></p>
 
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Purification of proteins: A-alpha

Piotr, Emilia

  1. pET15b+A_alpha in Rosetta strain from overnight culture inoculated in fresh LB and cultured until OD=0.5.
  2. Culture was induced with different concentrations of IPTG at 22°C and 37°C.
  3. Samples were collected twice: after 3h and next day (samples were centrifuged and frozen).

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