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Optimal annealing temperature


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#1 izzybusydizzy

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Posted 15 March 2010 - 02:55 PM

I am looking it equations that help me calculate the optimal annealing temperature to use in PCR. I have a lot of primer pairs to test coming up and was looking not for an easy way out but to undertand more if it is possible to get close to the correct annealing temperature and guide my gradient PCR's a little more.

I am reading the following paper:

Optimization of the annealing temperature for DNA
amplification in vitro
W.Rychlik*, W.J.Spencer' and R.E.Rhoads
Department of Biochemistry, Medical Sciences Building, University of Kentucky, Lexington, KY 40536
and 1The Perkin-Elmer Corporation, Northpointe Business Park, Fairfield, OH 45014, USA
Received April 10, 1990; Revised and Accepted August 22, 1990

In this paper they show the following equation:
In order to calculate Ta0PT, we developed the empirical formulation:

Taopt = 0.3Tmprimer + 0.7Tmproduct - 14.9

in which TmPrimer is the calculated Tm of the less stable primer-template
pair and TmProduct is the Tm of the PCR
product.


I have been pulling my hair trying to understand what they mean by "the less stable primer-template pair" Does anybody know? Please help!

#2 Adrian K

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Posted 16 March 2010 - 08:51 AM

Usually I will use the formula Tm= 2(A/T) + 4(G/C)
It just give the near and almost range ~+-5C
There will be no such thing as calculated optimal Tm, as the temperature is due to your setting, consumables and type of reagent and concentrations of your primers. There is no short cut, you have to do gradient all one by one.

Just my 2 cents

Adrian

Edited by adrian kohsf, 16 March 2010 - 08:52 AM.

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#3 izzybusydizzy

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Posted 16 March 2010 - 09:52 AM

Thank you Adrian.


I am still hoping to hear from some one if they know or understand what "less stable primer-template pair" means.




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