Team:Warsaw/Calendar-Main/19 June 2008

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<h3>Preparation of constructs: OmpA_alpha and OmpA_omega</h3>
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<h4>Michał K.</h4>
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<p> Inoculation of separate transformant colonies from plates with ligations: <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a> <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega>pACYC177+OmpA_omega</a> (liquid LB + kanamycin).</p>
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<p>Primers for fusion of protein A and ompA and alpha or omega part of beta-lactamase were designed:</p>
 
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<p>alphaL+link14+homo (alphaL_link)<br> CAGTGGTGGAAGCGGTGGC GGAGGATCTGGAGGAGGTGGTTCTGGAGGTGGAGGT CACCCAGAAACGCTGGTGAAAG<br>
 
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alphaP+STOP+XbaI+BamHI (alphaP_XB)<br> GG GGATCC GG TCTAGA TTA TTCGCCAGTTAATAGTTTGCG<br>
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<h3> Blue/white and rifampicin test #2 </h3>
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<h4>Piotr, Weronika</h4>
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omegaL+link14+homo (omegaL_link)<br> CAGTGGTGGAAGCGGTGGC GGAGGATCTGGAGGAGGTGGTTCTGGAGGTGGAGGT CTACTTACTCTAGCTTCCCGG<br>
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<ol>
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<li>The analysis of lacZ sequence from <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pZC320>pZC320</a>:<br>
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omegaP+STOP+EcoRI+PstI+BamHI (omegaP_EPB)<br> AAGGATCCAACTGCAGAAGAATTC TTACCAATGCTTAATCAGTG<br>
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There is no mutation in beta-galactosidase gene. Why are the colonies white?</li>
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<li>Colony <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a>.<br>
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OmpAL+NdeI (ompaL_N)<br> CATATGAAAGCTACTAAACTGGTACTGGG<br>
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OmpAP+link8+SacI+NotI+homo (ompaP_link)<br> GCCACCGCTTCCACCACT GCGGCCGC CA GAGCTC CCTCCTCCAGAACCTCC GCCGTTGTCCGGACGAGTGCCGATG</p>
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Template: DNA isolated from white colonies<br>
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Primers: <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#pZCseqL">pZCseqL</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#pZCseqR">pZCseqR</a></ol><br>
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<br>
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<table id="result">
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<tr><th colspan="3">Colony PCR program</th></tr>
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<tr><th>Temperature</th><th>Time</th><th>No. of cycles</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</td><td>0:45</td></tr>
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<tr><td>72&deg;C</td><td>1:30</td></tr>
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<tr><td>72&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>
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Latest revision as of 16:39, 28 October 2008

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Preparation of constructs: OmpA_alpha and OmpA_omega

Michał K.

Inoculation of separate transformant colonies from plates with ligations: pACYC177+OmpA_alpha pACYC177+OmpA_omega (liquid LB + kanamycin).

Blue/white and rifampicin test #2

Piotr, Weronika

  1. The analysis of lacZ sequence from pZC320:
    There is no mutation in beta-galactosidase gene. Why are the colonies white?
  2. Colony PCR.
    Template: DNA isolated from white colonies
    Primers: pZCseqL and pZCseqR


Colony PCR program
TemperatureTimeNo. of cycles
94°C4:00
94°C0:3028 cycles
48°C0:45
72°C1:30
72°C10:00
4°Cinfinite

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