Purification of proteins: A-alpha, Z-alpha and Z-omega
Piotr

A-alfa, Z-alfa and Z-omega from overnight culture were inoculated in fresh LB, cultured until OD=0,5 and then induced with different concentrations of IPTG in 22C and 37C. Samples were collected twice: after 3h and next day (samples were centrifuged and frozen)

Mutagenesis of protein A
Paweł

Mutagenesis of protein A was performed using 2 pairs of primers: ADelL+KpnI and ADelP (deletion of aminoacids involved in interaction with protein Z) and AMutL+KpnI and AMutP (changing of few aminoacids involved in interaction with protein Z).

Mutagenesis were performed on 3 vectors: pACYClac+ompA-A-omega, pACYClac+ompa-A-alfa and pACYClac+ ompa-alfa-A.

Each mutagenesis were performed using each primer at final concentration 0,1 uM, dNTPs at final concentration 0,25 uM and Walk (Pwo) polymerase (shipped by A&A Biotechnology), with 100 ng of DNA template.

PCR program (15 cycles):

94C 5 min

94C 30 s
55C 30 s
72C 10 min

72C 8 min

4C pause