Team:Warsaw/Calendar-Main/3 June 2008

From 2008.igem.org

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<h3>Preparation of T7 constructs</h3>
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<h3>Preparation of pMPMT5-AID+AIDT7 construct</h3>
<h4>Michał K.:</h4>
<h4>Michał K.:</h4>
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<ol>
<li>DNA (PCR products from previous day) gel electrophoresis and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">isolation</a> of DNA from proper band. </li>
<li>DNA (PCR products from previous day) gel electrophoresis and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">isolation</a> of DNA from proper band. </li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">Digest</a> of DNA with HindIII and SalI (2x Tango buffer).</li>
 
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">Digest</a> of pMPM-T5 + AID with HindIII and SalI.</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 reaction products.</li>
 
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of p.4 products (pMPM-T5 + AID transcription fusion with AID-T7 RNA-polymerase translation fusion).</li>
 
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <i>E.coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with ligation product.</li>
 
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<li>Plating transformants on LB+tetracycline.</li>
 
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">Digest</a> of pMPM-T5 + transcription fusion with EcoRHI and HindIII.</li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">Digest</a> of pMPM-T5 + transcription fusion with EcoRHI and HindIII.</li>
<li>Use of T4 polymerase for blunting.</li>
<li>Use of T4 polymerase for blunting.</li>

Revision as of 17:54, 5 October 2008

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Preparation of pMPMT5-AID+AIDT7 construct

Michał K.:

  1. DNA (PCR products from previous day) gel electrophoresis and isolation of DNA from proper band.
  2. Digest of pMPM-T5 + transcription fusion with EcoRHI and HindIII.
  3. Use of T4 polymerase for blunting.
  4. DNA gel electrophoresis.
  5. Isolation of DNA from proper band.
  6. Ligation of product p.11 (pMPM-T5 + T7 RNA-polymerase).
  7. Transformation of E.coli TOP10 with ligation product.
  8. Plating transformants on LB + tetracycline.

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