FAQ: Do I need to make any changes to the cycling conditions for my NGS library amplification I have previously been using with Phusion High-Fidelity PCR Master Mix?

Yes. Cycling conditions for NEBNext Q5 Hot Start HiFi PCR Master Mix differ from those recommended for Phusion High-Fidelity PCR Master. NEBNext Q5 Hot Start HiFi PCR Master Mix has been optimized for robust, low-bias amplification of NGS libraries. We recommend using an extension temperature of 65C for this application. Please consult the guidelines included in the NEBNext kit product manuals for more specifics.