Dear all
I am doing a droplet digital PCR with fluoro-oil like FC-40 and HFE-7500 supplemented by 5% EA surfactant(RAN). For enzymes like the HS Taq(NEB), I could get the positive droplets(all bright when the template DNA concentrated enough). Because I always get false positive droplet with NEB HS Taq, I stop to use other enzymes, like the Takara probe enzyme mix.
But somehow, these enzymes does not work in the droplet at all. All droplets, including those with enough template, are dark. I have ajusted the annealing temperature, but no matter how low the annealing temperature, all droplets are still dark.
Could anyone please tell my the mechanisms why these enzymes failed in the ddPCR? And if possible, could you please advise some solutions for this problem?
Thanks
Binbin
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