Annealing vs melting Ts for real time PCR primers - (Jun/15/2006 )
Hi, when choosing a primer for real time pcr, one wants a melting T between 58 - 60 degrees, and the cycles are run with a 60 degree annealing Temperature. Oligo 6 also returns an annealing temperature for the primers - do I also want the annealing Temp to be 58 - 60 deg?
If I have a primer with a lower annealing Temp (say 52 deg, which I've been using for standard PCR), and a 60 deg annealing Temp on the real time instrument, then I'm less likely to get priming because the T is too high, correct? I'm hoping to be able to use the primers I already have, rather then design new ones.
Can I set the annealing temp on the real time instrument to 52 deg? Or is this not recommended because of an increased likelihood of non-specific priming? Or is this ok as long as the annealing T of the primers is also 52 deg?
By far the best thing to do, if you have a real-time instrument that can run temperature gradients, is to set up a T gradient with a few dilution series of your reactions, and empirically see which temperatures work best. From the dilution series, you can calculate the efficiencies of your primers for each temperature. It's a quick and easy way to ensure that you're using the correct temperature for your primers, with your reagents, in your instrument.