Purification of proteins: Z-alpha and Z-omega

Piotr

Measurement of concentration of proteins (BCA method).

Cloning of protein A DNA to GeneArt plasmid

Antoni

Inoculation to liquid LB + ampicilin with bacteria carrying pGeneart+Z and pKS+A plasmids.

Cloning of alpha to pACYC177+OmpA_A_omega and pACYC177+OmpA_Z_omega

Michał K.

1. PCR on pUC19 plasmid using AlphaL+SacI and AlphaPlinkSac primers (gradient of annealing temperature from 55°C to 75°C; elongation length 60s; 35 cycles).
2. Gel electrophoresis and chioce of proper PCR conditions.
3. PCR on pUC19 plasmid using AlphaL+SacI and AlphaPlinkSac primers (annealing temperature - 65°C; elongation length 60s; 20 and 25 cycles (Fig. 1.)).
4. Gel electrophoresis and gel-out of 550 bp band.
5. Overnight digest of isolated PCR product with SacI (SacI buffer).