Suggest me a mastermix for conventinal PCR... - (Apr/11/2011 )
There is a huge advantage to using a standard mix of reagents when you are trying to optimize many things simultaneously. If you mix PCR with buffer, water, enzyme(s), dNTPs, primers, and template separately, you have many chances of things being contaminated, out of date, or lost. Standardizing everything except primers and template makes things much easier and consistent. You can do this yourself, of course.
I also basically don't optimize. Annealing is always at 55C, extension at 68C. The extension time is varied. This almost always works with well designed primers. If it doesn't, then I sometimes try a gradient PCR -- once. If that doesn't work I redesign primers, which almost always solves the problem.
I've never found it necessary to make up PCR reactions on ice, although I can see the theoretical reason why it might be necessary.
Since I was cloning last week I'd just note, that HotStarTaq is great on normal applications and direct sequencing of PCR products, but I wouldn't recommend it for PCR used for cloning. I had 1600 bp product that was faint, so I used 33 cycles (quite a lot, I know) and three out of four plasmids have at least one mutation.