Team:Warsaw/Calendar-Main/15 July 2008

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<h3>Cloning of protein Z DNA to OmpA constructs<br>
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Michał K.</h3>
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<h3>Cloning of protein Z DNA to OmpA constructs</h3><h4>Michał K.</h4>
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<p><ol><li> 4 colonies from transformation pACYC177+OmpA_Z_alpha were inoculated to liquid LB broth with kanamycin </li></ol></p>
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<p> Two colonies (<A href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-Z-omega>pACYC177+OmpA_Z_omega</a>) were inoculated to liquid LB with kanamycin.</p>
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<h3>Cloning omega-A fusion on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2B>pKS</a> (second attempt)</h3>
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<h4>Michał L., Ewa, Marcin</h4>
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<p><b><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcl">Polymerase Chain Ligation</a> on linker-A and omega-linker</b>
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<ul>
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<li>reisolated PCR product omega-linker - 4 µl<br>
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<li>reisolated PCR product linker-A - 13.5 µl<br>
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<li>primer
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaL+SacI">OmegaL+SacI</a> - 2 µl</li>
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<li>primer
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> - 2 µl</li>
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<li>Pfu buffer with Mg<sup>2+</sup> - 5 µl</li>
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<li>dNTPs - 1 µl</li>
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<li>H<sub>2</sub>o - 22 µl</li>
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<li>Program:</li>
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<ol>
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<li> 95&deg;C - 3'</li>
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<li> 95&deg;C - 30"</li>
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<li> 55&deg;C - 45"</li>
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<li> 68&deg;C - 1'</li>
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<li> go to step 2 25 x</li>
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<li> 68&deg; - 10'<br>
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<li> keep in 4&deg;</li>
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</ol></li>
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<li>gel electrophoresis of products</li></ul>
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</p>
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<h3>Cloning of protein A DNA to pET15b+OmpA-alfa plasmid in place of OmpA<br>Antoni</h3>
 
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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#plasmid_DNA_isolation">Isolation</a> of plasmids from bacterial cultures inoculated on previous day (pKS+A and pGeneart+Z). </li>
 
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<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of pKS+A and pGeneart+Z with SacI and NotI, <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing"> pGeneart dephosphorylation with CIAP</a>.</li>
 
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<li> Gel electrophoresis of digested plasmids 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  </li>
 
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<li> Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of pGeneart and A </li>
 
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</ol></p>
 
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<h3> Preparation of alpha+A conctruct</h3><h4>Antoni</h4>
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<p><ol><li>Gradient <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on alpha+A PCR products with <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaL+SacI">AlphaL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> primers.</li>
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<li>Gel electrophoresis. Again without satisfying results.
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<li>Third <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on alpha+A. This time two temperatures of annealing (68&deg;C and 72&deg;C) and gradient of DMSO.</li>
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<li>Gel electrophoresis (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/15_July_2008#fig1">Fig. 1.</a>).
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">Gel-out</a> of proper 1000 bp band.</li>
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</li></ol></p>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/c/c5/PCRalfa%2BAzDMSO.jpg"></a>
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<var><b>Fig. 1.</b>Gradient PCR on alpha+A products. Lanes 1-3, 5, 6 - annealing temperature 68&deg;C and DMSO concentration of 2%, 4%, 6%, 8% and 10% respectively. Lanes 7-11 - 72&deg;C and DMSO concentration increasing in the same maner. Lane 4 and 12 GeneRuler DNA Ladder Mix 1 &mu;g and 0.5 &mu;g respectively. 
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Latest revision as of 17:59, 26 October 2008

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Cloning of protein Z DNA to OmpA constructs

Michał K.

Two colonies (pACYC177+OmpA_Z_omega) were inoculated to liquid LB with kanamycin.

Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin

Polymerase Chain Ligation on linker-A and omega-linker

  • reisolated PCR product omega-linker - 4 µl
  • reisolated PCR product linker-A - 13.5 µl
  • primer OmegaL+SacI - 2 µl
  • primer AP+NotI - 2 µl
  • Pfu buffer with Mg2+ - 5 µl
  • dNTPs - 1 µl
  • H2o - 22 µl
  • Program:
    1. 95°C - 3'
    2. 95°C - 30"
    3. 55°C - 45"
    4. 68°C - 1'
    5. go to step 2 25 x
    6. 68° - 10'
    7. keep in 4°
  • gel electrophoresis of products


Preparation of alpha+A conctruct

Antoni

  1. Gradient PCR on alpha+A PCR products with AlphaL+SacI and AP+NotI primers.
  2. Gel electrophoresis. Again without satisfying results.
  3. Third PCR on alpha+A. This time two temperatures of annealing (68°C and 72°C) and gradient of DMSO.
  4. Gel electrophoresis (Fig. 1.).
  5. Gel-out of proper 1000 bp band.

Fig. 1.Gradient PCR on alpha+A products. Lanes 1-3, 5, 6 - annealing temperature 68°C and DMSO concentration of 2%, 4%, 6%, 8% and 10% respectively. Lanes 7-11 - 72°C and DMSO concentration increasing in the same maner. Lane 4 and 12 GeneRuler DNA Ladder Mix 1 μg and 0.5 μg respectively.

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