Team:Warsaw/Calendar-Main/30 September 2008

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Mutagenesis of protein A

Paweł

Results of sequencing: unfortunately all sequences where wild-type.

Preparation of BioBricks

Michał K.

  1. Isolation of HQs(??????) plasmids.
  2. Digest of isolated plasmids with EcoRI and BcuI (BamHI buffer).
  3. Digest of pACYC177+OmpA_omega_deltaA_alpha with BamHI and PstI (BamHI buffer).
  4. Dephosphorylation (CIAP) of plasmids.
  5. Gel elctrophoresis and gel-out of proper band: ??????.
  6. Digest of omega_link fragment (PCR from previous day) with EcoRI and BcuI (BamHI buffer)
  7. Digest of OmpA_omega with BglII and PstI (Orange buffer).
  8. Clean-up of above digest reactions.
  9. Overnight digest of purified pLac_OmpA_omega fragment (from previous day) with EcoRI (EcoRI buffer). DNA ends blunting with Klenow fragment.

Piotr

  1. E. coli TOP10 transformation with overnight ligations (RFP(??????) + Z and RFP(????) + OmpA_link).
  2. Plating on LB + ampicillin.
  3. Inoculation of bacterias received from iGEM HQs ( ).