Purification of proteins: A-alpha, Z-alpha and Z-omega
Piotr
- A-alpha, Z-alpha and Z-omega from overnight culture inoculated in fresh LB and cultured until OD=0,5
- Cultures 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-alpha and pACYClac+ ompa-alpha-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
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