# Optimum annealing temperature - Tm calculator (Aug/23/2007 )

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Dear all

i want to know how to determine the Optimum annealing temperature for my primers in order to run PCR

i know that it should be 5 degree less than the lower Tm of the 2 primers

but when i use the biomath progrom from promega site, i found three values for Tm

1- Basic Tm
2- Salt-adjusted Tm
3- Base-stacking calculated Tm

which one of them should i use to determine the Optimum annealing temperature as the three values are far different from each other

Best regards

-Jehane-

Try FastPCR program, it's free.

You can analyze your primers. You write them and the program give you the tm, the quality, if there are primer dimmer or hairsprings and the optimal annealing temperature for a PCR using that set of primers.

-aztecan princess-

QUOTE (aztecan princess @ Aug 23 2007, 06:27 AM)
Try FastPCR program, it's free.

You can analyze your primers. You write them and the program give you the tm, the quality, if there are primer dimmer or hairsprings and the optimal annealing temperature for a PCR using that set of primers.

COULD U PLEASE TELL ME BRIEFLY HOW TO GET OPTIMAL ANNEALING TEMPERATURE FROM THE FastPCR PROGRAM?????????

-Jehane-

First, had you installed the program?

If you have it already,

Open the program, select “Primer Test” from the list at the left.

Copy both primers sequence in the spaces at the left down and automatically will appear tm, CG%, Quality, dimmer and the optimal PCR annealing.

The value of quality describes thus the level of primer/PCR successfulness; this value varies from 120 for the “perfect & ideal” to 5 for the "worst" primer combination.

But as everything this is theory, you’ll may optimize experimentally.

-aztecan princess-

Thank u very much aztecan. it works with me very well. Best Regards.

-Jehane-

There are different formulae out there for calculating Tm, so saying the optimal annealing temperature is X degrees above or below a calculated Tm value will depend on the formula used. I think the only way to find out the optimal temperature is by doing a gradient PCR to get the empirical answer.

-wbla3335-

You’re welcome!! Jehane

As wbla3335 said, it’s better to do a gradient PCR, but specially, if you don’t have a gradient PCR machine, the program is very useful.

-aztecan princess-

i want to ask is there a special device for gradient PCR??? or any pce device could do it????

-Jehane-

QUOTE (Jehane @ Aug 24 2007, 01:09 PM)
i want to ask is there a special device for gradient PCR??? or any pce device could do it????

You need a gradient thermocycler, if you don’t have one, you need to run different PCR reaction testing different align temperatures in you regular thermocycler.

-aztecan princess-

How can i program the thermocycler to perform different reactions with different tempertures at the same time?????????????????????????????????????????????

-Jehane-

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