You can increase primer specificity by using touchdown PCR. For example, for GAPDH you could do touchdown PCR where the initial melting temperature is 72°C, and then with each cycle the melting temperature drops by one degree. After ten cycles the melting temperature will be set to 62°C; you then keep the melting temperature consistent at 62°C, and do a further 20 cycles.

Also, I don't know what polymerase you're using but a 1 minute extension time for an 81bp amplicon is longer than necessary.

Ciaran Daly Thank you for the reply. I may give touchdown a try. I agree 1min is a little overkill, it amplified better at a cooler temperature with fast PCR but the extra bands did not go away!

GAPDH has about 60 pseudogenes in the human genome so you may be picking up one of these in which case the primers are both binding to the correct sequence and different primers may be needed to get rid of the extra band

Hi Dania,

a reason for additional bands could also be contamination with genomic DNA - at least when your primer are spanning introns on the genomic level.

This can especially become a problem with long elongation time, when the polymerase has time to amplify larger amplicons. Maybe you should check for this (if you haven't done this yet)

For this reason I would avoud amplificationn times which greatly exceed the needed amplification time for your target amplicon. For the small amplicon 10 sec should be enough (15 sec if you want a somewhat longer amplification time) and for the longer one 15-20 sec should be enough.

If I see it correctly on the provided picture your annealing time is 1 min - you can also try do reduce the annealing time to 30 sec (or even 15 sec) - sometimes this improves specifity.