Team:Warsaw/Calendar-Main/4 June 2008

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<html><h3>Preparation of pMPMT5-AID+AIDT7 construct</h3>
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<html><h3>Preparation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID%2BAID-T7>pMPMT5-AID+AIDT7</a> construct</h3>
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<h4>Michał K.:</h4><ol>
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<h4>Michał K.</h4>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">Digest</a> of DNA fragment (AIDT7)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(2x Tango buffer), <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylation</a> (CIAP).</li>
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<p>
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<ol><li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">Digest</a> of DNA fragment  (AIDT7) 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 <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPM-T5 + AID</a> with HindIII and SalI(2x Tango buffer), <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylation</a> (CIAP).</li>
<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>
<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#ligation">Ligation</a> of purified DNA (pMPM-T5 + AID 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><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#electrotransform">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></ol></html><br>
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<li>Plating transformants on LB+tetracycline.</li></ol><br>
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<h3>Preparation of pMPMT5+T7 construct</h3>
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<h3>Preparation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-T7>pMPMT5+T7 </a>construct</h3>
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<h4>Piotr:</h4>
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<h4>Piotr</h4>
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<ol>
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<p>Inoculation of separate transformant colonies (liquid LB + tetracycline).</p>
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<li>Inoculation some separate transformant colonies (liquid LB + tetracycline).</li></ol>
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Preparation of pMPMT5-AID+AIDT7 construct

Michał K.

  1. Digest of DNA fragment (AIDT7) with HindIII and SalI (2x Tango buffer).
  2. Digest of pMPM-T5 + AID with HindIII and SalI(2x Tango buffer), dephosphorylation (CIAP).
  3. Clean-up of reaction products.
  4. Ligation of purified DNA (pMPM-T5 + AID with AID-T7 RNA-polymerase translation fusion).
  5. Transformation of E.coli TOP10 with ligation product.
  6. Plating transformants on LB+tetracycline.

Preparation of pMPMT5+T7 construct

Piotr

Inoculation of separate transformant colonies (liquid LB + tetracycline).

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