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 trunced A protein DNA fragment
Primers: AL+SacI AP+NotI

Elongation time: 30s

Annealing temperature: 60°C

20 cycles

3. Gel electrophoresis and isolation of 250 bp band.
4. Digestation of isolated PCR product, pACYC177+OmpA_alpha and pACYC177+OmpA_omega with NotI and SacI.
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.