Preparation of constructs with OmpA protein fusions

Michał K.

1. Isolation of plasmids from cultures inocluated on previous day.
2. Control digest of isolated plasmids with BamHI and NotI (BamHI buffer).
3. Gel electrophoresis (Fig. 1.) - we confirmed pACYC177 + OmpA_omega. We didn't obtain pACYC177 + OmpA_alpha probably because of a mistake in plating.
4. Ligation of pACYC177 and OmpA_alpha (1 hr).
5. Transformation of E. coli TOP10 strain with ligation products: pACYC177 and OmpA_alpha.
6. Transformants plating on LB + kanamycin.

Preparation of construct pKS with A protein

Michał L., Marcin

1. Inactivation of digestion enzymes and CIAP.
2. Ligation of digested PCR product and pKS for 2h at room temperature.
3. Transformation of E. coli TOP10 strain with 7 µl of ligation mix.
4. Transformants plating on LB + ampicillin + X-gal + IPTG.

Cloning of protein Z DNA to pET15b-OmpA-alpha in place of OmpA

Piotr, Antoni

1. Result of ligation of pET15b-OmpA-alpha with protein Z DNA: 16 colonies grown.
2. Each colony cultured overnight in LB + ampicilin.