Team:Warsaw/Calendar-Main/12 July 2008

From 2008.igem.org

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<h3>Preparation of constructs with OmpA protein fusions''' and '''Cloning of protein Z DNA to OmpA constructs</h3>
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<p><ol>
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<li> Isolation of plasmids from cultures inocluated on previous day (pCACYC177 + OmpA_alpha and pACYC177+OmpA_Z_omega). </li>
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<li> Control digestation of isolated plasmids with BamHI and SacI (we found good clones for both ligations).</li>
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</ol></p>
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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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<h3>Linked PCR Omega-A</h3>
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<p>
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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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<ul>
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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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<li>reisolated PCR product PCR-omega - 4 µl<br>
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<br>
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<li>reisolated PCR product A-homo - 13.5 µl<br>
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<li>primer
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'''Linked PCR Omega-A'''<br>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaL+SacI_N">OmegaL+SacI_N</a> - 2 µl</li>
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<li>primer
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reisolated PCR product PCR-omega - 4 µl<br>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI_N">AP+NotI_N</a> - 2 µl</li>
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reisolated PCR product A-homo - 13.5 µl<br>
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<li>Pfu buffer with Mg2+ - 5 µl</li>
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primer <html>
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<li>dNTPs - 1 µl</li>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaL+SacI_N">OmegaL+SacI_N</a></html> - 2 µl<br>
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<li>H2o - 22 µl</li>
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primer <html>
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<li>Program:</li>
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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>
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<ol>
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Pfu buffer with Mg2+ - 5 µl<br>
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<li> 95&deg;C - 3'</li>
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dNTPs - 1 µl<br>
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<li> 95&deg;C - 30"</li>
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H2o - 22 µl<br>
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<li> 55&deg;C - 45"</li>
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<br>
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<li> 68&deg;C - 1'</li>
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program:<br>
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<li> go to step 2 25 x</li>
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<br>
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<li> 68&deg; - 10'<br>
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1. 95&deg;C - 3'<br>
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<li> keep in 4&deg;</li>
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2. 95&deg;C - 30"<br>
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</ol></li>
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3. 55&deg;C - 45"<br>
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<li>gel electrophoresis</li></ul>
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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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</p>
</p>
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</html>
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Revision as of 17:53, 1 October 2008

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Preparation of constructs with OmpA protein fusions''' and '''Cloning of protein Z DNA to OmpA constructs

  1. Isolation of plasmids from cultures inocluated on previous day (pCACYC177 + OmpA_alpha and pACYC177+OmpA_Z_omega).
  2. Control digestation of isolated plasmids with BamHI and SacI (we found good clones for both ligations).

Linked PCR Omega-A

  • reisolated PCR product PCR-omega - 4 µl
  • reisolated PCR product A-homo - 13.5 µl
  • primer OmegaL+SacI_N - 2 µl
  • primer AP+NotI_N - 2 µl
  • Pfu buffer with Mg2+ - 5 µl
  • dNTPs - 1 µl
  • H2o - 22 µl
  • Program:
    1. 95°C - 3'
    2. 95°C - 30"
    3. 55°C - 45"
    4. 68°C - 1'
    5. go to step 2 25 x
    6. 68° - 10'
    7. keep in 4°
  • gel electrophoresis

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