Site-directed Mutagenesis - (Jun/30/2006 )
I have been doing the site-directed mutagenesis using the kit from Stratagene. Now I am trying to just buy Dpn1 and Pfu HiFidelity Polymerase and follow the same procedure. I have some questions:
Is it advisable to do PCR clean-up prior to adding Dpn1 as the enzyme is from Promega and the Pfu is from Stratagene?
Has anybody tried it that way?
I will greatly appreciate any help.
I don't do PCR cleanup before DpnI digestion, although the enzyme is from NEB and the Pfu from Stratagene. The enzyme is able to work in that buffer, therefore I don't feel like doing it because one never recovers 100% of what it's being cleaned
Cleanup is not necessary with Dpn I, although it probably is optimal.
Just use a bit more DpnI or let your reaction go on for a longer time and no purification is necessary (own experience and experience of colleagues).