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
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
Edited by Phanindra, 27 February 2014 - 12:27 PM.