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 (Polymerase Chain Ligation on linker-A and omega-linker

   Michał L., Ewa, Marcin

   • 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
   
   Fig. 1.Gradient PCR on alpha+A products. Lanes 1-3, 5, 6 - annealing temperature 68°C and DMSO concentration of 2%, 4%, 6%, 8% and 10% respectively. Lanes 7-11 - 72°C and DMSO concentration increasing in the same maner. Lane 4 and 12 GeneRuler DNA Ladder Mix 1 μg and 0.5 μg respectively.