pooling different pcr cycles? - (Dec/12/2010 )
If one is running low on template and needs more volume for PCR cleanup, can PCR products from different cycles be pooled together for example pool an 5 cycle PCR reaction with a 10 cycle PCR reaction? Say the PCR reactions were made from the same mastermix, only different cycles were done and both cycles gave very good product.
Would pooling the different cycles increase the final concentration of the pool or dilute it? And what would be the drawbacks from doing such a thing, besides risking more primer dimers in the final pool (which can be probably be cleaned up anyway).
well it depends if the plateau phase has been achieve after that many cycles.
If both PCR reaction mixes have reached the plateau phase than mixing the two could not change the concentration. Since the concentration of PCR product would be identical.
However if the plateau phase has not been achieved, then the reaction mix with the fewer number of cycles would logically has less product, and thus a lower concentration. Pooling the two would increase the amount of PCR product present but decrease the concentration.
I would add 5 cycle even 10 cycles is rather few. I use 25 cycle to amplify my PCR product.
Aside from not being as efficient as possible, I don't see a problem pooling the two.