Sub-optimal PCR - SOS (Mar/02/2005 )
I have a primer pair with Tm's 61.1 & 67.5 to amplify a target of 142 bp. I have tried touchdown PCRs, gradient PCRs according to either enzyme's manufacturers (Eppendorf Triple Master) or "lab-made" modifications. The thing is that my reaction is still not optimized, since I have been able to amplify this target inconsistenly in different scenarios. In terms of the quality of template DNA, it is good, as the amplification of 525, 700, 110 bp amplicons has been possible previously with those same samples. Does anybody has some recommendation?
Have you tried altering the Mg2+ and dNTP concentrations, often these are the determining factors in PCR, you can run gradients (with temperature gradient, if necessary) for each of these individually, which may help to optimise.
Other suggestions include replacing those reagents that can go "off" such as dNTPs and primers with too many freeze/thaw cycles.
DNA will also degrade with time and number of freeze/thaw cycles it has been through, if you have used this same tube of DNA for all your other PCRs, then it may have degraded. A suggestion is that once you extract DNA, aliquot it out into smaller batches, if you have enough to aliquot.