Team:Warsaw/Calendar-Main/30 September 2008

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Mutagenesis of protein A

Paweł

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

Preparation of BioBricks

Michał K.

  1. Isolation of pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids.
  2. Digest of isolated plasmids with EcoRI and BcuI (BamHI buffer) Fig. 1.
  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 pSB1A3 + Z(BBa_K103004) and pSB1A3 + OmpA_link).
  2. Plating on LB + ampicillin.
  3. Inoculation of bacteria received from iGEM HQs, carrying pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids.
Fig. 1. Control EcoRI/BcuI digests of isolated plasmids
1. Marker
2-3. digested plasmids
4. digested plasmid