Hi,
I am doing PCR with primers I designed using Primer 3 program. The first set gave me a great band in the right size. I have now moved to a second set and used a first set as a sort of positive control. Thus far, I have had the first set work with a very faint band and no bands in the second set and a case where neither worked. I have done multiple RNA isolations and RT-PCR to rule out the quality of my cDNA. I have been using a control for cDNA, beta actin, and that has been working perfectly with my failed primer attemps. Any suggestion

Did you change the annealing temperature for the second primer?

No, both sets of primers have a Tm of 60 or within 2 degrees of 60. Could my primers have gone bad? I only froze and unthawed them about 5x. Nothing is contaminated with buffer, dNTP, Taq, water, MgCl, because I used the same batch for my control b-actin and that worked perfectly

Although it is always best to minimise freezing and thawing by doing aliquots of your primers, I find it hard to believe that primer degradation actually occurred for just freezing and thawing 5x.

However did u use TE or water to dilute the primers? and did you mix the primers well after u diluted?

It could have been the case that you did not mix the primer well, preventing the solution from becoming homogenous.

Thats all i can say at this moment.

I thought it was strange that they would degrade so fast. I diluted them with water. As for mixing, I spin them down briefly and pipett up and down when making the PCR mix. Today I found out that the freezer I had been storing the primers aliquots and 100uM master solutions in a freezer that hotter than -20. When I asked they said they think it is around -2. I don't know if this could have led to a problem. I will try mixing more before I run any more PCRs. the control primers worked and I made those aliquots the same way I made the aliqouts for my designed primers. Any ideas help

Was the TAQ stored in -2 as well? If so that might be the problem