I'm looking for some advice on setting up a single-tube nested PCR assay.
The protocol I'm following has the first set of primers at a lower concentration
and higher annealing temperature. The annealing temperatures of my primers
are higher in the second round. Any idea if it is preferable to have the
first annealing temperature higher or not. Of course I could just try it
and see but any advice would be useful before I try.