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  • SunPenguin
    Member
    • Aug 2015
    • 38

    #1

    Best HiFi PCR enzymes for PreAmp?

    I know there have been some discussion about this before, but I want to bring it up again with a few specific concerns and see what people think.

    Typically, for preamplification, I've been using NEB's Phusion Flex hot-start enzyme. It's high fidelity, and it gives good yields in our hands. Recently though, I'm starting to worry about chimeric products, and a few people have been telling me Phusion is not a good choice for that. They invariably reference to Q5 or Kapa HiFi.

    There's a nature method paper (doi:10.1038/nmeth.1814) that also suggests that Kapa HiFi gives lower mutation rate, though looking around, some people seem to dispute this. I'm wondering if anyone else here have any input on this? I would of course hate to have to change to a new enzyme, but if there's a supposedly big difference...
  • nucacidhunter
    Jafar Jabbari
    • Jan 2013
    • 1250

    #2
    Kapa HiFi is only good when there are adapters at both ends of fragments and annealing temperature is high. In my experience it is not good for amplicon PCR when amplifying from a template. A 6 base microhomology within 10 base in 3’ end of a 25 base long primer is sufficient to prime and extend non-specific region if the last 3' base is complementary to template. This feature as Kapa calls it processivity can have down side.
    Last edited by nucacidhunter; 08-25-2015, 10:44 PM.

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