Cloning of omega_A DNA fragment to pACYC177+OmpA_alpha

Michał K.

1. Isolation of pKS+omega_A from culture inoculated on previous day.
2. Digest of pKS+omega_A with SacI and NotI (BamHI buffer).
3. Digestion (SacI and NotI) and dephosphorylation (CIAP) of pACYC177+OmpA_alpha.
4. Gel electrophoresis and gel-out of 4300 bp (pACYC177+OmpA_alpha) and 550 bp (omega_A) bands.
5. Ligation of DNA fragments from 2. and 4. (1 hr)
6. Transformation of E. coli TOP10 strain with ligation.
7. Transformants plating on LB + kanamycin.

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

1. Separate transformant colonies (transformation from previous day) inoculated to liquid LB with kanamycin.
2. Inoculation of pACYC177+OmpA_alpha and pACYC177+OmpA_omega - liquid LB with kanamycin.