Team:Warsaw/Calendar-Main/15 July 2008

From 2008.igem.org

(Difference between revisions)
Line 22: Line 22:
<li>reisolated PCR product linker-A - 13.5 µl<br>
<li>reisolated PCR product linker-A - 13.5 µl<br>
<li>primer  
<li>primer  
-
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaL+SacI_N">OmegaL+SacI_N</a> - 2 µl</li>
+
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaL+SacI">OmegaL+SacI</a> - 2 µl</li>
<li>primer  
<li>primer  
-
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI_N">AP+NotI_N</a> - 2 µl</li>
+
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> - 2 µl</li>
<li>Pfu buffer with Mg<sup>2+</sup> - 5 µl</li>
<li>Pfu buffer with Mg<sup>2+</sup> - 5 µl</li>
<li>dNTPs - 1 µl</li>
<li>dNTPs - 1 µl</li>

Revision as of 17:07, 11 October 2008

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Cloning of protein Z DNA to OmpA constructs

Michał K.

Two colonies (pACYC177+OmpA_Z_omega) was inoculated to liquid LB with kanamycin.

Preparation of alfa+A conctruct

Antoni

  1. Gradient PCR on alpha+A
  2. PCR with gradient of DMSO on alpha+A
  3. gel electrophoresis

Polymerase Chain Ligation on linker-A and omega-linker

Michał L., Ewa, Marcin

  • reisolated PCR product omega-linker - 4 µl
  • reisolated PCR product linker-A - 13.5 µl
  • primer OmegaL+SacI - 2 µl
  • primer AP+NotI - 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 of products

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