When I use
https://tmcalculator.neb.com/#!/main
I often get this message with a recommended annealing temperature of as high as 64 degrees
"Why is this annealing temperature so high?
The annealing temperature for high fidelity DNA polymerases like Q5 will differ from that of Taq-based polymerases. Q5 is a different family of polymerase than Taq, accounting for some of the difference in temperature requirements; the buffer composition also favors higher Tm values. This calculator incorporates all this information to suggest an optimal annealing temperature. "
But when I use these high annealing temps, I get very very low PCR runs. When I lower them, more normal DNA concentrations are obtained from the PCR due to primer specificity.
Has anyone else had this experience?
thank you! - Amy J.
Annealing is a difficult concept. Raising the annealing temperature always gains because the template has less time to re anneal with itself but as the Ta for the primer is raised the number of places that the primer can anneal gets less and a more spescific product is obtained but at still higher temperatures less primer will anneal and the efficiency of the pcr drops until it fails due to one primer not annealing. Very high temperature annealing primers are often high gc and for annealing and amplifying high GC templates and these templates can self anneal and stop the enzyme extension.
Sometimes the primer has high Ta due to it being AT rich but very long so different parts of the primer anneal elsewhere than wanted so causing dirty pcrs
Many researchers routinely run TA gradients which often show that a lower temperature with its increased efficiency is still highly specific because GC is less common in the human genome and this gives you the best practical solution
thanks Paul! very informative. I don't work with the human genome though ;-)
This does not usually happen to me. I have done a number of temperature gradient optimizations, and it usually comes out ideal pretty much exactly at NEB's prediction. When there are non-specific products, even quite a bit higher can work well in my hands. I guess this just tells us that nothing beats experimental optimization in PCR.
yes some lucky people amplify out of plasmids and almost any annealing temperature should work because there are not many other places to anneal. There is probably some poor soul working on organisms in hot vents with much GC in their genome and problems getting good amplification.
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