Team:Warsaw/Calendar-Main/25 June 2008

From 2008.igem.org

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<h3>Preparation of pMPMT5-AID+AIDT7 construct</h3>
<h3>Preparation of pMPMT5-AID+AIDT7 construct</h3>
<p><ol>
<p><ol>
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<li> Digest 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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<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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<li> Clean-up of digested PCR product. </li>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of digested PCR product. </li>
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<li> Gel electrophoresis of digested pMPMT5-AID and gel-out of proper band (4800 bp)</li>
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<li> Gel electrophoresis of digested pMPMT5-AID 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 band (4800 bp)</li>
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<li> Overnight ligation of purified DNA fragments</li></ol>
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<li> Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of purified DNA fragments</li></ol>
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  Michał L. Marcin and Ewa</h3>
  Michał L. Marcin and Ewa</h3>
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<p>We've ran gradient PCR to obtain Alpha-link and link-A. <br>Annealing temp 48&deg;C - 55&deg;C.<br>
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<ol>
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  Elongation time 1 - 2 h.</p>
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<li>We've ran gradient <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> to obtain Alpha-link and link-A. <br>Annealing temp 48&deg;C - 55&deg;C.<br>
 +
  Elongation time 1 - 2 h.</li>
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<p>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. </p>
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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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Revision as of 00:24, 5 October 2008

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Preparation of pMPMT5-AID+AIDT7 construct

  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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