Team:Warsaw/Calendar-Main/12 July 2008

From 2008.igem.org

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<h3>Preparation of construct <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII+A>pKS with A protein</a></h3> <h4>Michał L., Ewa, Marcin</h4>
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<p><ol>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation of plasmid DNA</a> from cultures inocluated on previous day. </li>
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<li> Control <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated plasmid with SacI and NotI (BamHI buffer) - 2 h.</li>
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<li> Gel electrophoresis of digested DNA.</li>
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<li> We are glad to find the proper clone and inoculate liquid LB with ampicillin to freeze bacteria carrying <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII+A>pKS-A</a> construct.</li></ol>
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<p>'''Preparation of constructs with OmpA protein fusions''' and '''Cloning of protein Z DNA to OmpA constructs'''<br>
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1. Isolation of plasmids from cultures inocluated on previous day (pCACYC177 + OmpA_alpha and pACYC177+OmpA_Z_omega). <br>
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2. Control digestation of isolated plasmids with BamHI and SacI (we found good clones for both ligations).
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<p>'''Linked PCR Omega-A'''<br></p>
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reisolated PCR product PCR-omega - 4 µl<br>
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reisolated PCR product A-homo - 13.5 µl<br>
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primer <html>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaL+SacI_N">OmegaL+SacI_N</a> - 2 µl<br>
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primer <html>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI_N">AP+NotI_N</a> - 2 µl<br>
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Pfu buffer with Mg2+ - 5 µl<br>
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dNTPs - 1 µl<br>
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H2o - 22 µl<br>
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<br>
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program:<br>
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<br>
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1. 95&deg;C - 3'<br>
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2. 95&deg;C - 30"<br>
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3. 55&deg;C - 45"<br>
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4. 68&deg;C - 1'<br>
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5. go to step 2 25 x<br>
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6. 68&deg; - 10'<br>
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7. keep in 4&deg;<br>
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<br>
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gel electrophoresis<br>
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<h3>Cloning of protein Z DNA to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-alpha>pET15b-OmpA-alpha</a> in place of OmpA</h3>
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<h4>Piotr, Antoni</h4>
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<p><ol>
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<li>Control <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated plasmids with NdeI and NotI. Two positives obtained (proper band: ~200 bp visible).</li>
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<li>One of positive plasmids transformed into <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> and plated on LB+amp, overnight, for further isolation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BZ-alpha>pET15b+Z+alpha</a> vector.</li>
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</ol></p>
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<h3>Preparation of constructs: OmpA_alpha and OmpA_omega #2</h3> <h4>Michał K.</h4>
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<p><ol>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation of plasmids</a> from cultures inocluated on previous day (<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pCACYC177 + OmpA_alpha</a>). </li>
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<li> Control
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated plasmids with BamHI and SacI (BamHI buffer).</li> <li> Gel electrophoresis - we found good clones (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/12_July_2008#fig1">Fig. 1.</a>).</li>
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</ol></p>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/2/28/Cztery.jpg" width=300 /></a><var><b>Fig. 1. </b>Control SacI/BamHI digests of plasmids which had a colony PCR product<br>
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1. Marker<br>
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2-4. digested plasmids which had a colony PCR product<br></var>
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Latest revision as of 17:46, 26 October 2008

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Preparation of construct pKS with A protein

Michał L., Ewa, Marcin

  1. Isolation of plasmid DNA from cultures inocluated on previous day.
  2. Control digest of isolated plasmid with SacI and NotI (BamHI buffer) - 2 h.
  3. Gel electrophoresis of digested DNA.
  4. We are glad to find the proper clone and inoculate liquid LB with ampicillin to freeze bacteria carrying pKS-A construct.

Cloning of protein Z DNA to pET15b-OmpA-alpha in place of OmpA

Piotr, Antoni

  1. Control digest of isolated plasmids with NdeI and NotI. Two positives obtained (proper band: ~200 bp visible).
  2. One of positive plasmids transformed into TOP10 and plated on LB+amp, overnight, for further isolation of pET15b+Z+alpha vector.

Preparation of constructs: OmpA_alpha and OmpA_omega #2

Michał K.

  1. Isolation of plasmids from cultures inocluated on previous day (pCACYC177 + OmpA_alpha).
  2. Control digest of isolated plasmids with BamHI and SacI (BamHI buffer).
  3. Gel electrophoresis - we found good clones (Fig. 1.).

Fig. 1. Control SacI/BamHI digests of plasmids which had a colony PCR product
1. Marker
2-4. digested plasmids which had a colony PCR product