Design or check a PCR primer

Aim for 18–25 bases, 40–60% GC, and a pair whose melting temperatures are within about 2 °C of each other. The 3′ end matters more than the rest: one or two G or C bases in the last five anchors it enough to extend, while four or five makes it stable enough to tolerate a mismatch further back and prime somewhere you did not intend. Avoid runs of four or more identical bases, which slip during synthesis.

Then check that the primer is available to the template at all. A primer that pairs with a copy of itself, or folds back on its own 3′ end, is consumed before it ever finds the target — and self-dimers amplify, which is what a smear at the bottom of a gel often is.

The melting temperature itself is where people get caught. Three models are in common use and they disagree by more than ten degrees on the same oligo: the Wallace counting rule assumes 1 M salt and no length term, the GC formula ignores sequence order entirely, and nearest-neighbour thermodynamics — what suppliers quote — accounts for both. A Tm is not a property of a sequence alone. It depends on the model, the salt and the oligo concentration, so quoting one without them is quoting a number nobody can reproduce.