The NEBNext Ultra II Q5 Master Mix is the most recent formulation of Q5 DNA polymerase that has been optimized for robust, high-fidelity amplification of next-generation sequencing (NGS) libraries. This new formulation further improves the uniformity of amplification of libraries, including superior performance with GC-rich regions. The polymerase component of the master mix, Q5 High-Fidelity DNA Polymerase, is a novel thermostable DNA polymerase that possesses 3´- 5´ exonuclease activity, and is fused to a processivity-enhancing Sso7d domain. Q5 also has the highest fidelity available (> 280-fold higher than that of Taq DNA Polymerase), resulting in ultra-low error rates. The NEBNext Ultra II Q5 Master Mix is an aptamer-based hot start formulation that allows simple room temperature reaction set up. The convenient 2X master mix format contains dNTPs, Mg++ and a proprietary buffer, and requires only the addition of primers and DNA template for robust amplification.