How do I correct a frameshift mutation? - (Jul/10/2007 )
I need to correct a frameshift mutation in a gene before cloning it into my target plasmid and have no idea how to go about it - can anyone recommend a protocol and/or useful background information?
Thanks in advance for any help offered!
look up site directed mutagenesis.
Here you have a primer set containing your DNA change. Using said primers, the plasmid is PCR amplifed. THis new plasmid formed, now contains the DNA change and the old plasmid is destroyed by DpnI digestion.
I agree - site directed mutagenesis is the way to go. You have 2 major options that I'm aware of:
1) Quick-change site directed mutagenesis with, for example, the kit from stratagene (pfu turbo polymerase). See http://www.stratagene.com/manuals/200518.pdf Instead of ordering primers that have a mutated base, you order primers with the desired base inserted.
2) Inverse pcr based site directed mutagenesis, as with the phusion enzyme. See http://www.neb.com/nebecomm/ManualFiles/manualF-541.pdf for illustrative pictures and protocol.
These options are fundamentally different - the first relies on linear amplification of the desired product, and digestion of unwanted template with DpnI, while the second method offers exponential amplification of the product, but requires a blunt end ligation (which works really well with the invitrogen ligase kits that have PEG in the buffer).