Cloning of protein Z DNA to OmpA constructs

Michał K.

Two colonies (pACYC177+OmpA_Z_omega) was inoculated to liquid LB with kanamycin.

Preparation of alpha+A conctruct

Antoni

1. Gradient PCR on alpha+A PCR products with AlphaL+SacI and AP+NotI primers.
2. Gel electrophoresis. Again without satisfying results.
3. Third PCR on alpha+A. This time two temperatures of annealing (68°C and 72°C) and gradient of DMSO.
4. Gel electrophoresis (Fig. 1).
5. Gel-out of proper 1000 bp band.

Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin

Polymerase Chain Ligation on linker-A and omega-linker

• reisolated PCR product omega-linker - 4 µl
  
• reisolated PCR product linker-A - 13.5 µl
  
• primer OmegaL+SacI - 2 µl
• primer AP+NotI - 2 µl
• Pfu buffer with Mg²⁺ - 5 µl
• dNTPs - 1 µl
• H₂o - 22 µl
• Program:
1. 95°C - 3'
2. 95°C - 30"
3. 55°C - 45"
4. 68°C - 1'
5. go to step 2 25 x
6. 68° - 10'
   
7. keep in 4°
• gel electrophoresis of products