MutD5 testing
Piotr
Inoculation MutD5 carrying: pACYC177+OmpA_Z_alpha, pACYC177+OmpA_Z_omega, pACYC177+OmpA_A_omega and pACYC177+OmpA_omega_ΔA to liquid LB + 0.25mM IPTG + kanamycin + 50 μl/ml + prey:
Mutagenesis of protein A
Paweł
- PNK phosporylation of mutagenesis products for 30 min at 37°C.
- PNK heat-inactivated by incubation at 75°C for 15 min.
- T4 ligase added and incubated for 1 h at room temperature.
- T4 ligase inactivated by incubation at 55°C for 20 min, then 5U of DpnI added and incubated at 37°C for 3 hours.
- After DpnI treatment mutagenesis products transformed into TOP10 and plated on LB + kanamycin plates.
Michał K.
- Isolation of plasmid from culture inoculated on previous day.
- Control digest of isolated plasmid with EcoRI and PstI (Orange buffer). No visible band on 250 bp. Fig. 1.
- Repettition of ligation of isolated DNA fragments of pSB1A3 and ΔA - overnight.
Fig. 1. Control EcoRI/PstIdigest of pSB1A3
1. Marker
2. digested pSB1A3