Team:Warsaw/Calendar-Main/20 May 2008

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<html><h3>Preparation of pMPMT5-AID+T7 and pMPMT5+AID-T7</h3>
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<html><h3>Preparation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAID%2BT7>pMPMT5-AID+T7</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAIDT7>pMPMT5+AID-T7</a></h3>
<h4>Michał K.</h4>
<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 PCR product for transcription fusion (T7 RNA-polymerase) 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 PCR product for <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAID%2BT7>transcriptional fusion</a> (T7 RNA-polymerase) 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 PCR product for translation fusion (AID+T7 RNA-polymerase) with NcoI and XbaI (1x 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 PCR product for <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAIDT7>translational fusion</a> (AID+T7 RNA-polymerase) with NcoI and XbaI (1x Tango buffer).</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 digested products from p.1 and p.2.</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 digested products from p.1 and p.2.</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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<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#digest">Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-omega>pMPM-T5</a> with NcoI and XbaI (1x 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#digest">Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-omega>pMPM-T5</a> with NcoI and XbaI (1x 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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<h3>Rifampicin test</h3>
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<h3>Rifampicin test #1</h3>
<h4>Michał L., Ewa, Marcin</h4>
<h4>Michał L., Ewa, Marcin</h4>
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<p> Plating colonies from the previous day on rifampicin.</p>
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<p> Plating cultures from the previous day on rifampicin.</p>
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Latest revision as of 14:02, 26 October 2008

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Preparation of pMPMT5-AID+T7 and pMPMT5+AID-T7

Michał K.

  1. Digest of PCR product for transcriptional fusion (T7 RNA-polymerase) with HindIII and SalI (2x Tango buffer).
  2. Digest of PCR product for translational fusion (AID+T7 RNA-polymerase) with NcoI and XbaI (1x Tango buffer).
  3. Clean-up of digested products from p.1 and p.2.
  4. Digest of pMPM-T5+AID with HindIII and SalI (2x Tango buffer), dephosphorylation (CIAP).
  5. Digest of pMPM-T5 with NcoI and XbaI (1x Tango buffer), dephosphorylation (CIAP).
  6. Gel-electrophresis and gel-out of proper bands (p.4 - 4850 bp, p.5 - 4250 bp).
  7. Electrophoresis to estimate the concentration of purified DNA.
  8. Ligation of purified products from p.1 and p.4.
  9. Electroporation of E. coli TOP10 with ligation product.
  10. Ligation of digested products from p.4 and p.5.
  11. Electroporation of E. coli TOP10 with ligation product.
  12. Transformants plating on LB + tetracycline.

Rifampicin test #1

Michał L., Ewa, Marcin

Plating cultures from the previous day on rifampicin.

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