Team:Warsaw/Calendar-Main/7 July 2008

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Preparation of constructs with OmpA protein fusions
Piotr:

  1. Digest of confirmed clones of pET15b+OmpA_alpha and pET15b+OmpA_omega with NdeI and BamHI.
  2. Digest of pACYC177 plasmid with NdeI and BamHI, dephosphorylation with CIAP.
  3. Gel electrophoresis of digested plasmids and gel-out of proper bands.
  4. Electrophoresis of gel-out products
  5. Overnight ligation of pACYC177 and OmpA_alpha.
  6. Overnight ligation of pACYC177 and OmpA_omega.

Preparation of construct pKS with A protein
Michał L., Marcin:

  1. Gradient PCR (achieving multiple copies of gene coding A protein):
    template DNA pDRIVE-TAPtag - 1 µl
    primer AP+NotI_N - 2 µl
    primer AL+SacI_N - 2 µl
    Pfu buffer with Mg2+ - 5 µl
    10 mM dNTPs - 1 µl
    Pfu Turbo polymerase - 0.5 µl
    H2O - 38.5 µl

    Program:
    1. 94°C, 3 min
    2. 94°C, 30 sec
    3. 62 to 74°C, 45 sec
    4. 72°C, 45 sec
    5. Repeat of elongation step 25X
    6. 72°C, 10 min
    7. Hold at 4 °C
  2. Gel electrophoresis of PCR product.
  3. Isolation of proper band (470 bp) from the gel.
  4. Overnight digest of isolated PCR product and pKS vector with NotI and SacI; to the reaction mix with pKS added 0.5 µl of CIAP.