8 replies to this topic

[celvas]

Hi all,

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?
Thanks

Edited by celvas, 13 October 2009 - 05:47 PM.

[microgirl]

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.

[celvas]

Thanks,
but what is the physico-chemical reason by which the annealing temperatures are so high.

[Superman]

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!

[fishdoc]

Superman, on Oct 24 2009, 05:21 PM, said:

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!

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.co.../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.

[swanny]

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.

[Prep!]

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!!! )

[Prep!]

http://www.premierbiosoft.com/tech_notes/P...mer_Design.html

might help too!!

[Superman]

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.

Cheers,
Superman

Edited by Superman, 26 October 2009 - 11:11 AM.