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PCR enzyme activity-can you do 50 cycles - (Oct/23/2006 )

Just a basic question about PCR enzymes, does anyone have a feeling for how many cycles your Taq polymerase will last in a standard PCR reaction? I always felt enzyme activity was exhausted after about 35 cycles, what do you all think? Can you just add more enzyme/primers/dNTPs to the initial reaction or is it necessary to do two-rounds of PCR?


The greatest number of cycles we have used is 45. It is bisulfite genomic sequencing PCR. We use short cycling time such as 10" for denaturing, 15-20" for annealing and extension.


you main problem is build up of pyrophosphate and dUTP in the reaction mix. These compounds inhibit the polymerase. Adding more/large PCR reaction mixes don't really help, as they aren't usually exhausted at this point.

The polymerase on the other hand is very stable, Taq has a half life of 3hrs at 94C. I don't think Taq denaturation would be a major problem at 35 cycles - 2.5hr?