Will ligase buffer affect polymerase fidelity?
Posted 22 June 2009 - 01:02 PM
My PCR conditions are:
50 ul reaction
0.5 uM of each primer
1.5 ul DMSO
1x HF buffer
1 ul of the ligase reaction as template
don't remember concentration of dNTPs, but pretty standard concentration
water to 50 ul
The point mutations really didn't affect my product, 3 were in non-coding regions of the sequence, and the 4th was in the wobble position, and didn't change the amino acid, but it's bothersome that there are that many mistakes in such a short sequence. The only thing I can think of is that the ligase (T4 from NEB) buffer somehow is affecting the fidelity of the polymerase, even though it's essentially a 1/50x concentration of the buffer in the PCR tube.
Posted 22 June 2009 - 01:25 PM
Posted 23 June 2009 - 05:38 PM
Anyway, the answer would be that the ligase buffer doesn't affect the polymerase.
I will keep the DMSO Phusion in mind for any future problems, though.