PCR - (Feb/27/2014 )

I am making point mutations in a membrane protein. All of the primers have a Tm of 80+. I have tried gradient PCR from 53-59 and also 60-70. I dont see any bands on the gel. I tried two step PCR by joining annealing and extension at 70. No success. The previous person in the lab also had the same kind of issues with hihn Tm (80+) but had success with annealing temparature of 55. I followed the same protocol and didnt work. have been using pfu Ultra. Any suggestions?

95C- 5 min

95C - 45 s

55, 53-59, 60-70C - 45s

68C - 10 min

extra step of 68 - 5min

4C- hold

TWO STEP PCR - 70 C annealing and extension for 12 min since pfu ultra takes more time

Dont know what more can I take care of

I take it this is some version of "round the horn SDM" or Qickchangea?  If so - the PCR is not an amplification reaction as such, it is mostly there to copy the plasmid and incorporate the mutation.  So, you PCRs might well have worked...