Although both master mixes contain Q5 Hot Start High-Fidelity DNA Polymerase, the master mix composition is different. NEBNext Ultra II Q5 Master Mix has been specifically optimized to faithfully amplify NGS libraries with the least amount of amplification bias. Additionally, the NEBNext Ultra II Q5 Master Mix does not form a precipitate. In contrast, NEBNext Q5 Hot Start HiFi PCR Master Mix sometimes forms a precipitate and requires additional mixing. The recommended number of PCR cycles are different for each formulation and it is important that the correct cycling guidelines are followed.