F primer has Tm around 70deg, while R-primer's Tm is 43deg. I am planning on doing a gradient PCR- not sure what is the lowest value to set for annealing, without risking non-specific amplification?
Also, would you recommend keeping the duration of annealing the same for the different temperatures?
Are you really stuck with those as the primers? If so, good luck to you, but my first reaction to this would be redesign if you can. Otherwise try 42 (lower Tm - 1) and see what you get. If you REALLY REALLY are stuck with this pair there are a couple of alternatives you can try if 42 gives you garbage. Honestly, I'm worried that the extension temp of your Taq is going to melt off the lower melting primer.
Hi, I would also suggest you to design new primers.
Try this program f.g.: http://frodo.wi.mit.edu/
Cheers, Nadine
Thank you, Guy and Nadine, for your suggestions. Yes, I will try and see if I can get an alternate primer for the lower Tm-primer.
Meanwhile, Guy - your answer made me think of another thing- general concept related to PCR.. Please forgive the naivete of this question- I'm wondering in case of a typical PCR, annealing temperature is between 50-60, and extension is at 72. And that works for most amplifications..
So your concern Guy- is it because the Tm of the reverse primer is in 40s..?
Thank you again!
Yeah, 40's is odd. If at all possible the mimum I like to use is 55C.
Usually the extension temperature doesn't knock the primers off (clearly), but with such a low Tm I would wonder. Shoot for greater GC content or longer or both if you can. Ending with a G or C on the 3' end (often called a "GC clamp") is usually a good idea too.
if you cannot design a new set of primer, try performing a touch down PCR
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