PCR - Taq dies toos soon? (Feb/27/2002 )
Hi I'm doing RT-PCR. P32 labeled it works fine with 21-25 cycles. I'm now trying to get it working unlabeled but it doesn't work very well. After 30 cycles I don't see any more amlification in my samples when I go up more cycles. I think the limiting factor is the lifetime of the Taq polymerse (I'm using from Roche).
I now improved my results by shortening the cycles from ca. 4min to ca. 2min but I don't want to go lower. Since I'm analysing large number of samples adding new Taq after a number of cycles is no option.
There are other DNA polymerases for PCR like Vent and Pfu does any one know if they have a longer lifetime and has anyone experience with this problem.
Is your dNTP more enough for your pcr?
In a general way, the Taqase(like that form promega) don`t go bad in such enviroment.
I use taq in 35 cycles PCR and it usually works well. I think you should add more dNTPs as the first suggestion or perfrom hot start PCR. It might help you.
This is an issue of the kinetics of the PCR reaction itself. Usually, the reagents like dNTPs and primers are not the limiting factor since a tiny fraction is incorporated into your products. Use 200uM EACH dNTP and 300-400nM primers which is 15-20pmols in a 50ul reaction. 10-50ng template is sufficient depending upon the size. 2.5U of Taq is plenty in 50ul. Now, vary the amount of MgCl2 from 1.0 to 2.5 mM in 0.5mM steps and perform your reaction. If you need more info Perkin-Elmer can steer you in the best direction with references.