How to determine PCR Annealing temperature for primers with different Tm - (Aug/17/2006 )

Hi there,

I'm relatively new to this - just a quick question.. I want to do a PCR and my forward primer has a Tm of 59.1 and reverse primer Tm is 74.3.

How do you go about determining what the best annealing temperature should be?

Many thanks!

Find someone with a gradient PCR machine and set a reasonable range of temperatures.

74.3 !!!! That is higher than the polymerase extension temperatures I've seen, the highest being 74 so far. I would redesign primers to be a more reasonable temperture and closer. 59.1 to 74.3 is a big jump. Also try 2-step PCR. No annealing temp just denature and extend.

Thanks!

But what extension temperature would you choose for the two-step PCR?

The one your enzyme requires

Why two step and not three?

94 deg C for the denaturation
Variable for the annealing
72 deg C for elongation if you're using standard Taq.

If you can't get the use of a gradient PCR machine then use one degree below your lower temp (i.e. about 58 deg C).

One piece of advice is when you design primers try to get the predicted Tms as close as possible. It's not that hard to design primers by eye that have Tms of only 2-4 deg C difference. I do this all the time. I don't bother with these bits of software.

Just try one of these different annealing temp. 55, 60 and 65C, one of them will work.

hi ...

I think u shud have gone through the primer design rules when u had made ur primers...which says only a 5° difference between the Tm of forward and reverse...but right now i think u can work out with temperatures in the range of 60s provided there is gradient pcr ,which will work....