PCR amplification of a promoter sequence for gel extraction worked beautifully using Phusion HF enzyme with GC (higher error but less picky) buffer. However, DNA concentration from the gel extraction was too low, so I need to do it again. Now with all the reaction parameters the same it is not working. So far I am using the same Phusion enzyme stock and buffers, the same primers which worked before, the same proportions, and the same thermo-cylcer set up. I have extracted fresh genomic DNA to amplify from thinking maybe the DNA had degraded with no luck. I make a mastermix fresh before each reaction by combining the Phusion enzyme, nuclease free water, dNTPs from a 10mM stock, and the GC buffer mix. Any ideas would be appreciated.