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a question about the annealing temperature - (Aug/09/2011 )

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?

-mengxibio-

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-

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

-Adrian K-

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 68C then, if I remember right (never tried it out so far myself).

-hobglobin-

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-

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


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

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.

-Trof-

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.

-Adrian K-