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annealing temperature for 3 primer pairs? - (Feb/15/2012 )

Dear all
I am a beginner to the field of real-time PCR, I have primers for three genes. the Tm of different forward and reverse primers for these genes are as follows:
gene A: For 55.4, Rev 57.8
geneB: For 56.6 , Rev 54.9
geneC: For 55.4, Rev 57.5
what would be the most suitable annealing temperature for setting the cycling conditions if I want to analyze these 3 genes simultaneously on the same plate by real-time PCR?
one more thing, If I established a calibration curve with a primer pair of one of these genes, can I use this calibration curve for the other 2 genes?
thanks a lot


You'll need to determine the best annealing temperature empirically. I'd suggest starting with each primer pair individually, to find a temperature that they work at (by gradient PCR), then try combining them.

Also keep in mind that you will need to check that running these reactions together won't cause them to out-compete each other for reagents.

I assume you are also using specific probes so that you can determine the level of each gene? If so, you will also need to titrate out the concentrations of these to find the appropriate value. And don't forget colour compensation.

As for the calibration curve, I think you will need one for each gene of interest.

If I were you, I'd go and do a LOT of reading about setting up and optimising multiplex reactions before attempting to do yours. You could also consider contacting the technical specialist at the company your machine is from, and the machine manual, as they should be able to help you work all of this out.

Good luck


If you're doing SYBR you can't. You need to have 3 different probes as leelee said. I wouldn't go into more than duplex anyway, especially if you're a beginner.


Yes it is SYBER green, so what would be the optimal temperature for each gene to start with?


There are different methods to calculate Tm. Your Tm looks quite low. Try to put yout primers to Primer3 Plus (select Primer Check) and see what Tm calculates there, I usually use annealing around 4-5 deg lower than this.
But the primers should be designed to have Tm (as calculated by primer3) around 65 so you can use 60 as annealing.

Calibration curve for each primer pair must be done with that primer pair.

And you can have different SYBR green assays on one plate, if they use same program (same annealing, elongation) I was mislead by previous post, that's a different thing, sorry.