Team:Warsaw/Calendar-Main/8 July 2008

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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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<p>
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<ol>
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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 ligation from previous day. </li>
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<li> Transformants plating on LB + kanamycin.</li></ol></p>
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<br>
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<p>'''Preparation of constructs with OmpA protein fusions'''<br>
 
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1. Transformation of E. coli TOP10  strain with ligation from previous day <br>
 
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2. Transformants plating on LB + kanamycin</p>
 
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<p>'''Preparation of construct pKS with A protein''' <br>
 
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1. Inactivation of digestion enzymes and CIAP<br>
 
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2. Ligation of digested PCR product and pKS 2h at room temperature<br>
 
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3. Transformation of E. coli TOP10 strain with 7 µl of ligation mix<br>
 
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4. Transformants plating on LB + ampicillin + X-gal + IPTG<br>
 
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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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<ol>
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<li>Gel electrophoresis of PCR products.</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><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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<br/>
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<table id="result">
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<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><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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</table>
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<br>
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<table id="result">
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<tr><th colspan="3"><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcl">PCL</a> program for omega-A fusion</th></tr>
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<tr><th>Temperature</th><th>Time</th></tr>
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<tr><td>94&deg;C</td><td>4:00</td></tr>
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<tr><td>94&deg;C</td><td>0:30</td><td rowspan="3">28 cycles</td></tr>
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<tr><td>48&deg;C-58&deg;C</td><td>0:45</td></tr>
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<tr><td>68&deg;C</td><td>2:00</td></tr>
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<tr><td>68&deg;C</td><td>10:00</td></tr>
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<tr><td>4&deg;C</td><td>infinite</td></tr>
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</table>
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<br/></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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</ol>
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</html>
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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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