Site directed mutagenesis-PCR - PCR reaction failing (Jan/06/2010 )
I need some help understanding site directed mutagenesis and any hints of what I may be doing wrong.
I have a sound protocal, so ive been told, that should work.
Overlapping primers to introduce the mutation using plasmid DNA as the template. My problem is that I my PCR reaction doesnt appear to be working. I run 10ul of a 50ul reaction on an agarose gel post reaction along side and equivilent amount of template in water. My template control shows a band but my reaction doesnt?
Ive been down reduced amounts of template and adding DMSO can help aid the reaction. Is there anything obvious that may help/what sort of concentrations of DMSO is usually required.
Ive been working on this way too long and cant figure out why it isnt working?? If anyone can help I can provide reaction details ect to pinpoint where im going wrong =)
It's a pcr reaction that is failing, so the normal pcr issues arise. Most likely the primers have problems, but it could be template quality, extension time, extension temperature, annealing temperature. Anything unusual about your template? Check primers for primer dimer and hairpin formation. Make sure they fully overlap. Make sure you are doing sufficient cycles (for example, ignore the instructions about only 15-18 cycles until this works). Make sure extension times are sufficient. Are you using the correct enzyme?