a question about the annealing temperature - (Aug/10/2011 )

I have seen some protocols about q-pcr.
I find that the primer and sample are same,but the annealing temperature of q-pcr and standard pcr are not same.
And the q-pcr is higher than standard pcr.
why do the q-pcr have a higher annealing temperature?

There is one widely used qPCR annealing temperature and that is 60 °C. Usually you design primers and then find optimal annealing temperature, in real-time PCR you design primers to work at 60. I'm not really sure what is the reason, but as this is higher that the classical starting annealing temperature (55 °C) it may be because the increased need for specificity. Higher annealing is more specific and DNA stays better denatured. I design all my "classic" PCR primers for 60 now, and I really love the results, clean and efficient.

Trof ,is it still acceptable if the temperature up to 63C ??

you can be even more higher and then make a two-step PCR with a step of simultaneous annealing and extension. The temps should be around 68°C then, if I remember right (never tried it out so far myself).

Yeah, Brilliant Dr H!
I should have think of that... my gradient have yet to reach the temperature... ok I will try later in the lab.
Cheers!

Adrian

Adrian K on Thu Aug 11 18:14:27 2011 said:

From the point of PCR yes, we have one assay using 63C annealing (and of course it's also possible to use lower temperatures if your product is specific). But with a probe-based system you have to consider, that Tm of probes is usually about 10C higher than the primers. If you increase annealing temperature too much it may be a problem with a probe binding, I would guess.

Hi All, I just try my new assay today... I try up to 66C.... eliminated the unspecific binding seen by naked eyes.
But I still yet to get rid of primer dimmer problem... I don't have probes. the assay is mean for Evagreen.