Cloning of omega_A DNA fragment to pACYC177+OmpA_alpha

Michał K.

Separate transformant colonies (tranformation of ligation of pACYC177+OmpA_alpha and omega_A from previous day) inoculated to liquid LB with kanamycin.

Cloning of truncated fragment of protein A

Michał K.

1. Optimization of PCR to obtain truncated fragment of protein A DNA.
   Primers: AL+SacI AP+NotI
   Elongation time: 30s
   - Optimization of annealing temperature (gradient from 55°C to 75°C)
   - Optimization of number of cycles(15, 20, 25, 30, 35)
2. PCR to obtain truncated A protein DNA fragment.
   Primers: AL+SacI AP+NotI
   Elongation time: 30s
   Annealing temperature: 60°C
   20 cycles
3. Gel electrophoresis and gel-out of 250 bp band.
4. Digest of isolated PCR product, pACYC177+OmpA_alpha and pACYC177+OmpA_omega with NotI and SacI (BamHI bauffer). pACYC177 vectors were also dephosphorylated.
5. Clean-up of digestion reaction.
6. Gel electrophoresis for estimation of DNA concentration.
7. Overnight ligation: pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA.