Team:Warsaw/Calendar-Main/20 May 2008

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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>
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<p>Michał K: <br>
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<h4>Michał K.</h4>
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1.<html><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> - translation fusion: AID + T7 RNA-polymerase - optimalization (temperature gradient 60&deg;C - 80&deg;C).</p>
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<p><ol>
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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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<p>Primers: </p>
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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>
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<p><html>
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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 products from p.1 and p.2.</li>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDlNrH">AIDlNrH</a> and  
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7pXbSal">T7pXbSal</a></html> <br>
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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>
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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-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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Template DNA: purified PCR products from 16 May - AID and T7 RNA-polymerase for translation fusion <br>
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<li>  Gel-electrophresis 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 bands (p.4 - 4850 bp, p.5 - 4250 bp).</li>
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<li>Electrophoresis to estimate the concentration of purified DNA.</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 products from p.1 and p.4.</li>
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Annealing time: 4 minutes <br>
 
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35 cycles <br>
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<li>  <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#electrotransform">Electroporation</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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2. Optimalization of <html><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> - translation fusion: AID + T7 RNA-polymerase - MgCl2 cocentration and number of cycles.
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of digested products from p.4 and p.5.</li>
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<p>Primers: </p>
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<p><html>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#electrotransform">Electroporation</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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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDlNrH">AIDlNrH</a> and
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7pXbSal">T7pXbSal</a></html></p>
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<li>  Transformants plating on LB + tetracycline.</li></ol>
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</p>
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<h3>Rifampicin test #1</h3>
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<h4>Michał L., Ewa, Marcin</h4>
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<p> Plating cultures from the previous day on rifampicin.</p>
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</html>
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Template DNA: purified PCR products from 16 May - AID and T7 RNA-polymerase for translation fusion <br>
 
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Annealing temperature: 73&deg;C<br>
 
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Annealing time: 4 minutes <br>
 
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3.  Gel electrophoresis of PCR products.
 

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.