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 DNA

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 </li>

3. Gel electrophoresis and isolation of 250 bp band. </li>
4. Digest of isolated PCR product, pACYC177+OmpA_alpha and pACYC177+OmpA_omega with NotI and SacI. </li>
5. Clean-up of digestion reaction. </li>
6. Gel electrophoresis for estimation of DNA concentration. </li>
7. Overnight ligation: pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA.</li></ol> </html>