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
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
- if you provided the
- 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.