How to determine PCR annealing temperature - (Oct/27/2004 )
Hi, somebody knows how to estmate T annealing of a set of primers?
Hi, The simplest way is:
Tm = 2(A + T) + 4(C + G) degrees. It is roughly right. But lot of softwares calculate Tm for you.
That is T melting but I wanted T annealing!!
Right, that is Tm. Annealing temperature (Ta) is deduced from Tm, that is 5-10C below Tm.
Usually for standardising a pcr annealing temperature we start from a temp. 5 degrees lower than the Tm.
but the one that I'm doing now has an annealing temperature of 10 degrees higher than the Tm ( since at lower annealing temp. I got many nonspecific products together with the desired one.)
The exact Tm depends on additional factors of course (like salt content etc.). As already stated it is a basic strategy to start PCRs 5 °C lower, than move your way up. If the annealing temperature needs to be very high (due to unspecifcs) one can e.g. add betain (or one of those solutions that often come with PCR reagents) to enhance the specifity at lower temperatures.
I have two primers
one has 340 bp Tm>100
one has 20 bp Tm=65.9
The PCR product shoule be around 850 bp.
What annealing temperature should I use?
What other suggestion for me, such as the time for extension?