I am using Q5 hot start DNA polymerase in RT-PCR and am able to generate good results, however, whenever i apply it in ddPCR i am not getting any results. I am aware of optimization, however, I beleive the issue is Q5 is not working in ddPCR, i would like to know if anyone else has the same experinece? Is there any way to use Q5 in ddPCR?
Many thanks in advance.
Hey Elar Ad
I have experienced that hot start PCR gives better reproducibility. So I suggest you to go through.
it will not work in case you want to use probes (no 5'-3' exo acitvity). Further there is a reference dye in the mix I assume (for drop calling) so I do not see the benefit of using the non standard mix
Sven
Hi Sven Reister,
I truly believe that KAPA HIFI (with 3' to 5' exo nuclease activity the same as Q5 hot start and no 5' to 3' exo activity) work in ddPCR and it has a great fidelity that standard mix which has no 3' to 5' exo activity does not have. High Fidelity is significant element here which reduce errors that standard polymerase can brigh about. I use both enzymes however, depends on the chemistry and complexity of the probes they behave differently.
I think you cannot used Q5 and other Hifi DNA polymerase enzyme in ddPCR.
Because, the normal pcr buffer cannot genarate droplet in ddPCR system.
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