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.