Overloading a PCR? - Is this possible? (Nov/18/2006 )

Hey all,

This is not specifically a real-time question. I was setting up just a regular PCR and someone told me not to add to much template because it can overload the reaction. IS this possible? I would think too much template would just make the reaction hit exponential phase faster and produce more product.

I could see this happeneing if you were doing real time and you just did not have enough beacons to report how much product was being made, but in a normal DNA-DNA endpoint PCR is this something to worry about?

Also I took physical chemistry, does anyone know of a good book or site that talks about the physical chemistry and kinetics of PCR?

Thanks
Neil Stewart

well, previously I thought so too, but it turned out to a big mistake to increase the template concentration where after the end of the PCR I did not find any amplification curve!! I could only obtain the a very nice amplification curve after reducing the template concentration to 1/80 the concentration I was using

The overload reaction can happens, definitively. If so, then the possible differences of expression between your samples could be misinterpreted. If you don't want diminish template, you can try with a semi-quantitative PCR assay, running samples of your reaction at different cycles number, i.e. 20, 25 and 30 cycles, to verify that the PCR is not saturated or overloaded.
Regards.

It's not only the amount of DNA added to consider... In some cases, when adding the DNA we are also adding contaminants that might inhibit the PCR reaction (i.e. RT buffer when adding cDNA, or SDS from digestion buffer when using gDNA that has not been cleaned up...). So diluting template also contributes to dilute contaminants...