Team:Warsaw/Calendar-Main/7 July 2008

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<h3>Preparation of constructs: OmpA_alpha and OmpA_omega #2</h3>
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<h3>Preparation of constructs with OmpA protein fusions<br>
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<h4>Piotr, Weronika</h4>
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Piotr:</h3>
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<p>It's the second attempt to clone OmpA_alpha and OmpA_omega to pACYC177, but now thank's to Paweł we've got both fusions on pET15b.</p>
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<p>
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<ol>
<ol>
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<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of confirmed clones of pET15b+OmpA_alpha and pET15b+OmpA_omega with NdeI and BamHI </li>
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<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of confirmed clones of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-alpha>pET15b+OmpA_alpha</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> with NdeI and BamHI (Tango 2x buffer). </li>
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<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of pACYC177 plasmid with NdeI and BamHI, <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylation with CIAP</a> </li>
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<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177>pACYC177</a> plasmid with NdeI and BamHI (Tango 2x buffer), <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">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> 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 (OmpA_alpha- 1000 bp, OmpA_omega - 800 bp). </li>
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<li>Electrophoresis of gel-out products</li>
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<li>Electrophoresis of gel-out products.</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 pACYC177 and OmpA_alpha </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 <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177>pACYC177</a> and OmpA_alpha. </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 pACYC177 and OmpA_omega </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 <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177>pACYC177</a> and OmpA_omega. </li>
</ol>
</ol>
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</p>
 
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<html><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> Gradient PCR (achieving multiple copies of gene coding A protein):<br>
 
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template DNA pDRIVE-TAPtag - 1 µl<br>
 
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primer
 
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI_N">AP+NotI_N</a></html> - 2 µl<br><html>
 
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primer
 
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI_N">AL+SacI_N</a> - 2 µl<br>
 
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Pfu buffer with Mg<sup>2+</sup> - 5 µl<br>
 
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10 mM dNTPs - 1 µl<br>
 
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Pfu Turbo polymerase - 0.5 µl<br>
 
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H2O - 38.5 µl<br>
 
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<br><html>
 
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Program:<br>
 
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1. 94&deg;C, 3 min<br>
 
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2. 94&deg;C, 30 sec<br>
 
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3. 62 to 74&deg;C, 45 sec<br>
 
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4. 72&deg;C, 45 sec<br>
 
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5. Repeat of elongation step 25X<br>
 
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6. 72&deg;C, 10 min<br>
 
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7. Hold at 4 &deg;C<br>
 
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</li>
 
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<li> Gel electrophoresis of PCR product</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">Isolation of proper band (470 bp) from the gel</a></li>
 
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<li> Overnight <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated PCR product and pKS vector with NotI and SacI; to the reaction mix with pKS added 0.5 µl of <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">CIAP</a>. </li>
 
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</ol>
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</p>
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<h3>Cloning omega-A fusion on pKS (second attempt)</h3>
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<h4>Michał L., Ewa, Marcin</h4>
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<p>We have just received corrected version of omegaP-link10-homo2 primer. Let's hope this time everything will be fine. We are repeating the PCRs:</p><br/>
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<table id="result">
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<tr ><th colspan="4"><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a></td></tr>
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<tr><th>Product</th><th>Template</th><th>Primers</th><th>Product length</th></tr>
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<tr><th>linker-A</th><td>pDRIVE-TapTag</td><td><a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+link10+homo2">AL+link10+homo2</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> </td><td>470 bp</td></tr>
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<tr><th>omega-linker</th><td><a href=http://www.fermentas.com/techinfo/nucleicacids/mappuc1819.htm>pUC19</a></td><td><a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaL+SacI">OmegaL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaP+link10+homo2">OmegaP+link10+homo2</a></td><td>400 bp</td></tr>
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</table>
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<br> <br>
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<table id="result">
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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>gradient 48-55&deg;C</td><td>0:45</td></tr>
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<tr><td>72&deg;C</td><td>0:50</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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Latest revision as of 16:33, 26 October 2008

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Preparation of constructs: OmpA_alpha and OmpA_omega #2

Piotr, Weronika

It's the second attempt to clone OmpA_alpha and OmpA_omega to pACYC177, but now thank's to Paweł we've got both fusions on pET15b.

  1. Digest of confirmed clones of pET15b+OmpA_alpha and pET15b+OmpA_omega with NdeI and BamHI (Tango 2x buffer).
  2. Digest of pACYC177 plasmid with NdeI and BamHI (Tango 2x buffer), dephosphorylation with CIAP.
  3. Gel electrophoresis of digested plasmids and gel-out of proper bands (OmpA_alpha- 1000 bp, OmpA_omega - 800 bp).
  4. Electrophoresis of gel-out products.
  5. Overnight ligation of pACYC177 and OmpA_alpha.
  6. Overnight ligation of pACYC177 and OmpA_omega.

Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin

We have just received corrected version of omegaP-link10-homo2 primer. Let's hope this time everything will be fine. We are repeating the PCRs:


PCR
ProductTemplatePrimersProduct length
linker-ApDRIVE-TapTagAL+link10+homo2 and AP+NotI 470 bp
omega-linkerpUC19OmegaL+SacI and OmegaP+link10+homo2400 bp


TemperatureTime
94°C4:00
94°C0:3028 cycles
gradient 48-55°C0:45
72°C0:50
72°C10:00
4°Cinfinite

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