2 replies to this topic

[oznyd]

Hi, my primer Forward primer Tm is 71.8 C and reverse primer Tm is 54.3 C, is this too much difference, will it be ok
so i would like to know how will i set the PCR temperature, i will use ex taq and taq polymerase

Thanking you

[HomeBrew]

Hello oznyd -- welcome to the BioForums!

Those Tm's are pretty far apart -- I usually shoot for a 2 - 3 degree difference between my primer pairs, and a Tm of around 60C. I don't know whether a set that differs by ~20 degrees will work, as I've never attempted it. How are you picking your primers? Can you select another forward primer closer to the reverse primer's Tm?

[swanny]

Does the high temp primer have additional sequence (restriction sites, GC clamps etc)? If so, ignore them for the initial Tm calculations, because those bases will not interact with your template.