Cloning of truncated fragment of protein A

Piotr

1. Isolation of plasmids from cultures inoculated on previous day.
2. Control digest with SacI and BamHI (BamHI buffer).
3. Gel electrophoresis - proper clones founded for both products of ligation.

Checking if OmpA_omega_A_alpha gives ampicillin resistance

Piotr

Inoculation OmpA_omega_A_alpha from various IPTG concentrations (in mmol/mL) (0, 0.1, 0.25, 0.5, 0.75, 1) into same IPTG concentrations, but with various ampicillin concentrations (in mmol/mL)(25, 50, 75, 100) in ratio: 1:50.

Inoculation of OmpA_omega_A_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75 mmol/mL (replication is necessary)

Checking OmpA_omega_A_alpha and OmpA_A_alpha expression

Piotr

1. Spinning
2. Suspending
3. Adding of lysis buffer
4. Boiling
5. Putting into poliacrylamide gel
6. Transfer onto nitrocellulose
7. Blocking
8. Anti-A antibody binding
9. Washing
10. Anti-rabbit antibody binding
11. Developing with BCIP and NBT

Michał L., Ewa, Marcin

Inoculation of pACYC177+OmpA_alpha and pACYC177+OmpA_omega - liquid LB with kanamycin.

Preparing pACYC177+OmpA_omega_deltaA construct

Michał K.

1. Transformation of E. coli TOP10 strain with ligation.
2. Transformants plating on LB + kanamycin.