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:

• Z_alpha for pACYC177+OmpA_Z_omega
• Z_omega for pACYC177+OmpA_Z_alpha
• A_apha for pACYC177+OmpA_A_omega
• A_alpha for pACYC177+OmpA_omega_ΔA

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 ΔA (BBa_K103003)

Michał K.

1. Isolation of plasmid from culture inoculated on previous day.
2. Control digest of isolated plasmid with EcoRI and PstI (Orange buffer) Fig. 1. No visible band on 250 bp.
3. Repettition of ligation of isolated DNA fragments of pSB1A3 and ΔA - overnight.