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.