Team:Warsaw/Calendar-Main/11 July 2008

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Preparation of constructs with OmpA protein fusions

  1. Colony PCR on colonies from plates with transformations OmpA_alpha.
  2. Confirmed transformant colonies inoculated to liquid LB with kanamycin.

Cloning of protein Z DNA to OmpA constructs

2 colonies was inoculated to liquid LB broth with kanamycin

Preparation of construct omega-A

  1. PCR A in 50 µl
    template DNA - pKS-A4 1 µl
    primer AP+NotI_N - 2 µl
    primer AL+link10+homo2_N - 2 µl
    Pfu polymerase buffer + Mg2+ (from Fermentas) - 5 µl
    dNTPs - 1 µl
    Pfu turbo - 0.5 µl
    H2o - 38.5 µl

    Program:
    1. 95°C 3'
    2. 95°C 30"
    3. 62°C 45"
    4. 72°C 45"
    5. 72°C 10'
    6. keeping in 4°C
  2. PCR omega in 50 µl
    template DNA - pUC19 1 µl
    primer OmegaLS - 2 µl
    primer AOmegaPli - 2 µl
    Pfu polymerase buffer + Mg2+ (from Fermentas) - 5 µl
    dNTPs - 1 µl
    Pfu turbo - 0.5 µl
    H2o - 38.5 µl
    Program:
    1. 95°C 3'
    2. 95°C 30"
    3. 62°C 45"
    4. 72°C 45"
    5. 72°C 10'
    6. keeping in 4°C
    25 cycles
  3. Gel electrophoresis
  4. Reisolation from agarose gel