Oczyszczanie białek: A-alfa, Z-alfa i Z-omega Piotr zaszczepienie A-alfa, Z-alfa i Z-omega z chodowli nocnej do świeżego LB, chodowanie do OD=0,5 indukcja rużnymi stężeniami IPG w 22 i 37C - pobranie próbek po 3h i następnego dnia rano (próbki zwirowano, odżucono pożywkę i zamrożono)

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