NEBNext® High-Fidelity 2X PCR Master Mix is the original non-hot start NEBNext formulation of Q5® High-Fidelity DNA Polymerase. This formulation is also included as a component in the original NEBNext standard workflow library prep kits for Illumina, and in NEBNext library prep kits for Ion Torrent.
However, the NEBNext Ultra II Q5 Master Mix (NEB #M0544) is now available, and this more recent formulation is a hot start and also further improves the uniformity of amplification of libraries, including superior performance with GC-rich regions. The NEBNext Ultra II Q5 Master Mix is also included as a component in all NEBNext Ultra II kits, for DNA and RNA library preparation.
NEBNext High-Fidelity 2X PCR Master Mix is the original non-hot start NEBNext formulation of Q5 DNA Polymerase. This formulation is also included as a component in the original NEBNext standard workflow library prep kits for Illumina, and in NEBNext library prep kits for Ion Torrent.
The NEBNext Ultra II Q5 Master Mix (NEB #M0544) is now available, which further improves the uniformity of amplification of libraries, including superior performance with GC-rich regions.
The polymerase component of both master mixes, 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.
NEBNext High-Fidelity 2X PCR Master Mix contains dNTPs, Mg++ and a proprietary buffer, and requires only the addition of primers and DNA template for robust amplification.
An E. coli strain that carries the Q5 High-Fidelity DNA Polymerase gene.
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To ensure optimal performance, the master mix should be thawed and resuspended prior to use. Stability testing using up to 20 freeze/thaw cycles has shown no negative effect on master mix performance. The NEBNext High-Fidelity 2X PCR Master Mix may be liquid at -20°C.
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Connelly, C.M., Porter, L.R. and TerMaat, J.R. (2020) PCR amplification of a triple-repeat genetic target directly from whole blood in 15 minutes as a proof-of-principle PCR study for direct sample analysis for a clinically relevant target BMC Med Genet; 15, 130. PubMedID: 25495904, DOI: 10.1186/s12881-014-0130-5
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