Primers annealing temperatures - (Oct/13/2009 )
I tried to figure out an answer in the web but I did not find anything. My question is: why in common PCR, for primer-template annealings, the temperature must be 50ºC or higher?
Not sure exactly what you mean, but the temps can be less than 50deg. However, the lower your annealing temp is the more likely you are to get non-specific amplification. The basic idea is to use the highest annealing temp you can and still get amplification of the fragment you want.
but what is the physico-chemical reason by which the annealing temperatures are so high.
How can you work out the highest annealing temp possible? I read that annealing should be between 3-5 degrees lower than the Tm of the primers. Does anyone know if this is true. Sucks if it is cos I have a PCR running right now with annealing at 63 degrees and primer Tm's are around 55 degrees.
I am having a PCR nightmare atm!
Superman on Oct 24 2009, 05:21 PM said:
I am having a PCR nightmare atm!
I think you can do 10 different calculations and come up with 10 different answers. Annealing will depend on a number of factors including salt concentration, and different enzymes have different salt concentrations in the buffer. I use Pfu UltraII frequently, and it recommends either 3 or 5 degrees below the melting temp. Phusion, on the other hand, recommends 3 degrees over the melting temp because of a higher salt concentration.
Bottom line is that both of these are generally guidelines, but not set rules. It's a place to begin, and optimize from there. I've often used annealing temps that were higher than the given melting temp. You use what works.
For calculating melting temps, I typically use IDT's Oligo Analyzer ( http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/ ), but if you notice on that page, there's a place to put in different concentrations for different PCR components... if you change those values, the annealing temps change, so as I said, the melting temp is a starting point, not an absolute.
Remember that the Tm is when 50% of the primer will bind, so you need to go below that by a few degrees to stabilise the primer-template interaction.
You don't want the annealing to happen at too low a temperature, mis-priming aside, because at those low temps, Taq doesn't work very well. My rule of thumb is that you need to be annealing at ~60-62 degrees for reasonable Taq activity. Once you have 5-10 bases addes, you can happilygo to 72, where taq works very well.
One consequence of having too low an annealing temp is that one or both primers will anneal to sequences other than the true target, as internal single-base mismatches or partial annealing may be tolerated. This can lead to nonspecific amplification and will consequently reduce the yield of the desired product if the 3′ -most base is paired with a target. Conversely, too high annealing temp may yield little product, as the likelihood of primer annealing is reduced.
Also the universal formula for calculation of annealing temp is
Ta Opt = 0.3 x(Tm of primer) + 0.7 x(Tm of product) - 25
•Tm of primer is the melting temperature of the less stable primer-template pair.
•Tm of product is the melting temperature of the PCR product.
(Got it from a site!!! )
might help too!!
Thanks for everyones help. Much appreciated.
PCR worked great today. I ran a temp gradient and found annealing to be best at 52-55 degrees. Keep having trouble with what looks like contamination aswell which sucks. Keep getting a nice perfect little band in all my negatives. Yesterday there was no band and today there is a band. Can't tell where the contamination is coming from. Should get to the bottom of it soon I hope.