Having problems with a RT-PCR reaction that works fine with regular Taq but gives absolutely no product with Phusion Hot Start II from Thermo. I replaced my dNTP stock with new material, tried different template. Still no luck so I'm scratching my head over this one. For what its worth the enzyme isn't that old either.
Phusion is derived from a polB-type polymerase, hence -unlike plain Taq- it cannot amplify templates containing dU (which is a common contaminant in some commercially available dNTP mixes). Try dNTPs from a different supplier.
You might have already tried this, but just in case. Perhaps you need to increase the amount of Mg2+ in the reaction mix. We use a final concentration of 2 mM, slightly higher than the concentration in the 1x buffer. Also, you might want to double check the annealing temperature of your primers, which is normally different from the temperature used for regular Taq. For Phusion, you can check:
https://www.neb.com/protocols/2012/09/06/protocol-for-phusion-hot-start-flex-dna-polymerase-m0535
Thank you both, Alejandro and Héctor. I'll look into both possibilites. I hope it's not the dNTPs as I just bought fresh from the same company that I got the enzyme (Thermo).
My experience might help. I had got my PCR band at 55 deg with normal Taq polymerase but same Band appeared on 72 deg when I used Phusion HS. so before start check your primer on Tm analyzer on thermo web site with phusion and taq buffer and use accordingly. This will solve your problem. Gudluck
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques