Preparation of constructs with OmpA protein fusions

1. Colony PCR on colonies from plates with transformations OmpA_alpha.
2. Confirmed transformant colonies inoculated to liquid LB with kanamycin.

Cloning of protein Z DNA to OmpA constructs

2 colonies was inoculated to liquid LB broth with kanamycin

Preparation of construct pKS with A protein

1. Colony PCR on colonies from plates with transformations pKS-A
   Primers used: AL+SacI and AP+NotI
2. Confirmed transformant colonies inoculated to liquid LB with ampicillin

Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin:

We had to start form scratch with this one.

1. PCR A in 50 µl
   template DNA - pKS-A4 1 µl
   primer AP+NotI_N - 2 µl
   primer AL+link10+homo2_N - 2 µl
   Pfu polymerase buffer + Mg²⁺ (from Fermentas) - 5 µl
   dNTPs - 1 µl
   Pfu turbo - 0.5 µl
   H2o - 38.5 µl
   
   Program:
   
   1. 95°C 3'
   2. 95°C 30"
   3. 62°C 45"
   4. 72°C 45"
   5. 72°C 10'
   6. keeping in 4°C
2. PCR omega in 50 µl
   template DNA - pUC19 1 µl
   primer OmegaLS - 2 µl
   primer AOmegaPli - 2 µl
   Pfu polymerase buffer + Mg²⁺ (from Fermentas) - 5 µl
   dNTPs - 1 µl
   Pfu turbo - 0.5 µl
   H2o - 38.5 µl
   Program:
   
   1. 95°C 3'
   2. 95°C 30"
   3. 62°C 45"
   4. 72°C 45"
   5. 72°C 10'
   6. keeping in 4°C
   25 cycles
3. Gel electrophoresis
4. Reisolation from agarose gel

Preparation of construct pKS with A protein

1. Isolation of plasmid DNA from cultures inocluated on previous day.
2. Control digest of isolated plasmid with SacI and NotI; 2 h
3. Gel electrophoresis of digested DNA
4. We are glad to find the proper clone ;-) and inoculate liquid LB with ampicillin to freeze bacteria carrying pKS-A construct