Question

i am using allele specific pcr assay to detect a polymorphism. my primers, that detect the mutant alleles, have 68 deg C Tm. To detect the normal allele I've designed a shorter primer; which obviously has a lower Tm. Is it ok to run all of these primers together even though the other primer is shorter & has a lower Tm?

Answer 1

As long as you are careful about the temp you run it at and are sure that's what you actually want for your results, I don't see why not.

Answer 2

i guess i was confused since everywhere i look it's emphasized that the primers should have similar/close Tm.

Answer 3

They should - when they are relatively close and I'm in a heck of a hurry, I've been known to run different primers at the same time. When the temps are quite different, it becomes tricky because the right temp for one primer is too low or high for the other one. In general, you're best served to do them separately.

Answer 4

u mean i should run the pcr to detect the normal allele separately?