Team:Warsaw/Calendar-Main/8 July 2008

From 2008.igem.org

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<h3>Preparation of constructs with OmpA protein fusions<br> Piotr</h3>
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<h3>Preparation of constructs: OmpA_alpha and OmpA_omega #2</h3>
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<h4>Piotr</h4>
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<h3>Cloning omega-A fusion on pKS (second attempt)</h3>
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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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<h4>Michał L., Ewa, Marcin</h4>
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<li>Gel electrophoresis of PCR products</li>
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<li>Gel electrophoresis of PCR products.</li>
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<li>Gel-out of proper bands</li>
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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 band.</li>
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<li>PCL reaction to create omega-A fusion:<br>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcl">PCL</a> reaction to create omega-A fusion:<br>
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<tr><th>Product</th><th>Templates</th><th>Primers</th><th>Product length</th></tr>
<tr><th>Product</th><th>Templates</th><th>Primers</th><th>Product length</th></tr>
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<tr><th>Omega-A fusion</th><td>Omega-linker + linker-A</td><td>OmegaL+SacI + AP+NotI </td><td>1440 bp</td></tr>
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<tr><th>Omega-A fusion</th><td>Omega-linker + linker-A</td><td><a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaL+SacI">OmegaL+SacI</a> + <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a></td><td>750 bp</td></tr>
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<br/></li>
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<li>Gel electrophoresis of PCL products</li>
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<li>Gel electrophoresis of PCL products.</li>
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<li>We have obtained no PCL product (weird?)</li>
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<li>We have obtained no PCL product (weird?).</li>
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<h3>Preparation of construct pKS with A protein<br>
 
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Michał L., Marcin:</h3>
 
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<p><ol>
 
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<li> Inactivation of digestion enzymes and CIAP.</li>
 
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of digested PCR product and pKS 2h at room temperature.</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> strain with 7 µl of ligation mix.</li>
 
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<li> Transformants plating on LB + ampicillin + X-gal + IPTG.</li></ol></p>
 
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Latest revision as of 16:33, 26 October 2008

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Preparation of constructs: OmpA_alpha and OmpA_omega #2

Piotr

  1. Transformation of E. coli TOP10 strain with ligation from previous day.
  2. Transformants plating on LB + kanamycin.


Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin

  1. Gel electrophoresis of PCR products.
  2. Gel-out of proper band.
  3. PCL reaction to create omega-A fusion:

    ProductTemplatesPrimersProduct length
    Omega-A fusionOmega-linker + linker-AOmegaL+SacI + AP+NotI750 bp

    PCL program for omega-A fusion
    TemperatureTime
    94°C4:00
    94°C0:3028 cycles
    48°C-58°C0:45
    68°C2:00
    68°C10:00
    4°Cinfinite

  4. Gel electrophoresis of PCL products.
  5. We have obtained no PCL product (weird?).

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