We synthesized overlapping primers (following QuikChange method), but in my lab we have Q5 enzyme.
A few days ago, we received a pair of primers we had sent to be synthesized for doing an amino acid substitution. Unfortunately we did it on SnapGene following QuikChange method, I mean fully overlapping except in the mutation, which is in the middle of the primers. I ran a couple of PCR using Q5 master mix 2X, but I didn't get amplification, just a smearing . I tried changing the concentration of primers, template, and 2-step PCR (because Ta=72°C). I used Tm calculator from NEB to check Tm and Ta, but I am not sure if it is correct, since it doesn't take into account mismatches with the template. Has anyone gone through something similar? I don't know if I am calculating the Ta in the right way. All the ideas/suggestions will be welcome! Thank you for reading.
F_F150Y: GCCAGCGCCTCTtcTCCAACCCGAGCATC
R_F150Y: GATGCTCGGGTTGGAgaAGAGGCGCTGGC
My director suggests changing primers, but before that, I would like to know WHY IS NOT WORKING :(