Quickchange site directed mutagenesis - Success with a 13 kb plasmid? (May/05/2004 )
Has anyone had success using the Dpn-I/Pfu method of introducing mutations? I'm trying to change 2 aa (3 bp change) that are 5 aa away from each other.
Any tips? I'm not using the kit... I plan to purchase the Pfu Ultra and DpnI seperately.
Hints/suggestions greatly appreciated!
It works brilliantly, even without the kit. The key is the design of the primers (use the guidelines in the stratagene manual, you can download it online) and their purity...it also helps to transform into a RecA- strain that's fairly competent (the Inoue method works fine). If you're paranoid about ensuring you're product is parental-DNA-free, you can use a 2 hour DpnI digestion. Other than that, it's a piece of cake.
The strategy we've used for designing primers for situations for yours is just using the web utility to design a single-mutation primer, and then adding the second mutation to that and elongating the primer on the appropriate side.
Thanks so much btavshan!
I had one more question... How big of a plasmid were you able to mutate? My plasmid is around 13kb and if I recall, stratgene's quickchange kit is for plasmids <8kb.
Also, did you ever encounter errors (i.e. pfu induced mutations?).