Team:Warsaw/Calendar-Main/12 July 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 digest of isolated plasmids with BamHI and SacI (we found good clones for both ligations).

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_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 of products