Team:Warsaw/Calendar-Main/25 June 2008

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<h3>Preparation of pMPMT5-AID+AIDT7 construct</h3>
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<h3>Preparation of pMPMT5-AID+AIDT7 construct<br>Michał K.</h3>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">Digest</a> of isolated PCR product (from previous day) and pMPMT5-AID with HindIII and XbaI, dephosphorylation of pMPMT5-AID with CIAP </li>
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">Digest</a> of isolated PCR product (from previous day) and pMPMT5-AID with HindIII and XbaI, dephosphorylation of pMPMT5-AID with CIAP </li>
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  Elongation time 1 - 2 h.</li>
  Elongation time 1 - 2 h.</li>
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<li>We have successfully amplified Alpha-link and link-A. Now its time for PCL (Polymerase Chain Ligation) to create fusions  Alpha-A and Omega-A. </li>
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<li>We have successfully amplified Alpha-link and link-A. Now its time for PCL (Polymerase Chain Ligation) to create fusions  Alpha-A and Omega-A. </li></ol>
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Revision as of 15:31, 9 October 2008

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Preparation of pMPMT5-AID+AIDT7 construct
Michał K.

  1. Digest of isolated PCR product (from previous day) and pMPMT5-AID with HindIII and XbaI, dephosphorylation of pMPMT5-AID with CIAP
  2. Clean-up of digested PCR product.
  3. Gel electrophoresis of digested pMPMT5-AID and gel-out of proper band (4800 bp)
  4. Overnight ligation of purified DNA fragments

PCR of Alpha-link and link-A
Michał L. Marcin and Ewa

  1. We've ran gradient PCR to obtain Alpha-link and link-A.
    Annealing temp 48°C - 55°C.
    Elongation time 1 - 2 h.
  2. We have successfully amplified Alpha-link and link-A. Now its time for PCL (Polymerase Chain Ligation) to create fusions Alpha-A and Omega-A.

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