Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

Doubled polymerase in the rxn, does this increase pcr efficiency? - (May/01/2014 )

I accidentally left a set-up PCR on the bench overnight. I then spiked in more polymerase and ran the PCR. The PCR was more efficient than expected, and I am wondering if the reason could be because of the polymerase and not because I had more starting material than I thought. 


The details are: 


Phusion HS II HF Polymerase. I put 0.5ul (1U) in originally and the next day I put in 0.5ul more (ie 1U more) into a 50ul rxn. 


This is important to me because it is for an RNA-seq library prep and the PCR looked over-enriched at 16 cycles. I still have half of my starting material and am not sure if I should enrich at 14 or 16 cycles again. The reason I am doing 16 cycles is because I started with a low amount of RNA in the very beginning ~500ng when my lab normally starts with ug's. 


The key to my inquiry though is will increasing the amount of polymerase (doubling) alter the efficiency of the PCR and make more product. 





The PCR efficiency is most affected by the component that is most limiting.


In a PCR with good setting, the limiting at the start of the reaction should be only the template. But of course if you put there less primers than needed, the primer concentration is soon limiting. The same with dNTPs etc.

The polymerase itself is rarely limiting at the start of the reaction, but that changes in time, because denaturation steps degrade it gradualy, so puting more polymerase (possibly to the one that was still partially OK) can result to more active polymerase at later cycles. This Phusion is a hot-start enzyme, so should be blocked and protected at lower temperatures.


But if you need to specifically tell if in your set up it made a notable difference, and don't waste the rest of material, probably the best would be take a similar amount of .. that template thing you have (not really sure what it means, RNA is not amplified) and try to run one PCR as normal and one exactly as you did before, left on the bench then with added enzyme. You can compare both on one gel to tell the difference.


Otherwise you probably can guess and chose a 15 cycles as a compromise..