Team:Warsaw/Calendar-Main/29 July 2008

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<h3>Checking if OmpA_omega_A_alpha gives ampicillin resistance<br>
 
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Piotr</h3>
 
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<p>Inoculation OmpA_omega_A_alpha from various IPTG concentrations (in mmol/mL) (0, 0.1, 0.25, 0.5, 0.75, 1) into same IPTG concentrations, but with various ampicillin concentrations (in mmol/mL)(25, 50, 75, 100) in ratio: 1:50.</p>
 
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<center><h4 style="text-align: center">Measurement of bacterial culture growth (OD) in the evening:</h4> </center>
 
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<table id="result" align="center">
 
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<tr><th rowspan="2">ampicillin concentration (μg/mL):</th><th colspan="6">IPTG concentration (mmol/mL):</td></tr>
 
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<tr><th>0</th><th>0.1</th><th>0.25</th><th>0.5</th><th>0.75</th><th>1</th></tr>
 
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<tr><th>25</th><td class="live">1.558</td><td class="live">1.469</td><td class="live">1.587</td><td class="live">1.49</td><td class="live">1.566</td><td class="live">1.311</td></tr>
 
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<tr><th>50</th><td class="live">1.425</td><td class="live">1.435</td><td class="live">1.524</td><td class="live">1.055</td><td class="live">0.920</td><td class="live">0.935</td></tr>
 
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<tr><th>75</th><td class="live">1.09</td><td class="live">0.989</td><td class="live">1.447</td><td class="live">0.971</td><td class="live">0.951</td><td class="live">0.992</td></tr>
 
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<tr><th>100</th><td class="live">0.09</td><td class="live">0.685</td><td class="live">1.378</td><td class="live">1.078</td><td class="live">0.977</td><td class="live">0.992</td></tr>
 
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</table>
 
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<p>
 
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Inoculation of OmpA_omega_A_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75 mmol/mL (replication is necessary)</p>
 
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<h3>Checking OmpA_omega_A_alpha and OmpA_A_alpha expression<br>
 
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Piotr</h3>
 
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<ol>
 
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<li>Spinning</li>
 
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<li>Suspending</li>
 
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<li>Adding of lysis buffer</li>
 
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<li>Boiling</li>
 
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<li>Putting into poliacrylamide gel</li>
 
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<li>Transfer onto nitrocellulose</li>
 
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<li>Blocking</li>
 
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<li>Anti-A antibody binding</li>
 
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<li>Washing</li>
 
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<li>Anti-rabbit antibody binding</li>
 
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<li>Developing with BCIP and NBT</li>
 
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</ol>
 
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[a photo of the gel is top be put here]
 
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<h4>Michał L., Ewa, Marcin</h4>
 
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<p>
 
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<ol>
 
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<li>Separate transformant colonies (transformation from previous day) inoculated to liquid LB with kanamycin. </li>
 
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<li>Inoculation of pACYC177+OmpA_alpha and pACYC177+OmpA_omega - liquid LB with kanamycin.</li></ol>
 
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</p>
 
</html>
</html>

Revision as of 21:44, 11 October 2008

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Cloning of omega_A DNA fragment to pACYC177+OmpA_alpha

Michał K.

  1. Isolation of pKS+omega_A from culture inoculated on previous day.
  2. Digest of pKS+omega_A with SacI and NotI (BamHI buffer).
  3. Digestion (SacI and NotI) and dephosphorylation (CIAP) of pACYC177+OmpA_alpha.
  4. Gel electrophoresis and gel-out of 4300 bp (pACYC177+OmpA_alpha) and 550 bp (omega_A) bands.
  5. Ligation of DNA fragments from 2. and 4. (1 hr)
  6. Transformation of E. coli TOP10 strain with ligation.
  7. Transformants plating on LB + kanamycin.