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Tm of primers - prediction and verification - (Mar/28/2012 )

Hi, for qPCR at our lab we use a StepOnePlus instrument and Power Sybr Green PCR 2x Master Mix, both from ABI. The standard cycling include 40 cycles of 15s at 95 degrees C and 1min at 60 for annealing/extension.

When we design our primers (Using primer3 or primer-blast), we normally intent that they have Tm of 60-65C (we don't specify neither nor ) , but when we receive the oligos (from Sigma), the calculated Tm is different. I guess this is because the algorithms that are used for Tm prediction are different.

Since I was not sure that 60C is the perfect annealing/extension temperature for efficiently amplifying the amplicons of the last 7 GOI I am studying, I ran various reactions in a temperature gradient and then for some GOI I got 54C as an optimal temperature, for others - 58, 62, 64.

Then I read that the real Tm of a primer (oligonucleotide) depends on various factors, but mostly on Mg and Na concentrations.

Therefore, it will be nice to hear some opinions on the following:

- when you design primers for qPCR with SybrGreen, do you provide the nor at your primer design software ? Is this information available for the Master Mix you're using (in ours it is not!)

- if you provided the and have you noticed if your qPCR efficiency is maximal when you use Tannealing 5C lower than Tm ? Most technical guides on qPCR advice on such temperature difference for optimal amplification.

- does somebody know is there a way to physically check/validate what is the real Tm of a given primer in its qPCR environment - inside a specific mix of Master Mix + H2O+ primers ?



No I don't and it's not.
I only check if efficiency is within acceptable limits, and optimize temperature only in case of dimers or so. Most of our qPCR assays run together with other assays, so same temperature is prefered.
I doubt that and IMHO it's not worth the effort, it's easier just to design new primers.