In PCR optimization, What is the best annealing temperature to prevent primer-dimer
I'm not sure that there is a blanket annealing temperature that works in all cases since the optimum annealing temperature of your locus varies with the length and GC content of the amplicon. Typically, higher temperatures are more stringent and reduce primer-dimer. I would advise running a gradient PCR with your locus on a positive control to determine your best annealing temperature. Does primer dimer form in both unknowns and positive controls? If so, you should reduce the primer concentration.
I agree with Misa about increasing the annealing temperatures for more specificity and the gradient. Further, I would suggest adding 5% DMSO if you are still getting primer dimers after reducing the primer concentration.
there is no standard annealing temperature (Ta) for all PCR reactions to prevent primer dimer. the easiest method to obtain TA is by using Ta below 2-5 oC of your primer's melting temperature (Tm). at least the difference of Tm of your forward and reverse primer is not significant. But, this guessing method sometime not work well.
In this way, you may to have to perform gradient PCR first to obtain the best Ta. may be you can start 5 oC below to above of your primers Tm.
Wish you luck.
- Badges
- Science method