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 plasmids from cultures inoculated on previous day.
2. Control digest of isolated plasmids with EcoRI and PstI (Orange buffer). No proper colonies found.