FAQ: My template is GC-rich or supercoiled. How can I optimize my product yield using Phusion® High-Fidelity DNA Polymerase?

* Add 2-8% DMSO in the final reaction. This often requires a reduction in annealing temperature by 2-5°C.
* Optimize initial denaturation time. For most templates the optimal initial denaturation time is 30 seconds, but up to 3 minutes can be used for complex templates.
* If HF buffer has failed, try using GC buffer. GC buffer allows better amplification of GC-rich templates, but slightly lowers fidelity.
Alternately, try Q5® High-Fidelity DNA Polymerase (NEB# M0491) and include the Q5 High GC Enhancer additive.