Team:Warsaw/Calendar-Main/23 September 2008

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MutD5 testing

Piotr

Inoculation MutD5 carrying: OmpA_Z_alpha, OmpA_Z_omega i OmpA_omega_A to liquid LB + 0.25mM IPTG + kanamycin + 50 μl/ml + prey (Z_alpha for OmpA_Z_omega, Z_omega for OmpA_Z_alpha and A_apha for OmpA_Z_alpha).

Mutagenesis of protein A

Paweł

  1. PNK phosporylation of mutagenesis products for 30 min at 37°C.
  2. PNK heat-inactivated by incubation at 75°C for 15 min.
  3. T4 ligase added and incubated for 1 h at room temperature.
  4. T4 ligase inactivated by incubation at 55°C for 20 min, then 5U of DpnI added and incubated at 37°C for 3 hours.
  5. After DpnI treatment mutagenesis products transformed into TOP10 and plated on LB + kanamycin plates.

Preparation of BioBricks

Michał K.

  1. Isolation of plasmid from culture inoculated on previous day.
  2. Control digest of isolated plasmid with EcoRI and PstI (Orange buffer). No visible band on 250 bp.
  3. Repettition of ligation of isolated DNA fragments of RFP(??????) and deltaA - overnight.


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