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  • NEBNext® High-Fidelity 2X PCR Master Mix

    Description






    The NEBNext High-Fidelity 2X PCR Master Mix is specifically optimized for the robust, high-fidelity amplification of next-generation sequencing (NGS) libraries, regardless of GC content. 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 High-Fidelity DNA Polymerase also has an ultra-low error rate (> 100-fold lower than that of Taq DNA Polymerase and 6-fold lower than that of Pyrococcus furiosus (Pfu) DNA Polymerase). The buffer component of the master mix has been optimized for robust amplification, even with GC-rich amplicons.

    M0541
    Figure 1: Comparative Analysis of Different DNA Polymerases with Known Low Coverage Regions.
    Indexed libraries were prepared from human IMR90 DNA and split into individual samples for library amplification. Amplification was performed using 8 cycles of PCR with Phusion High-Fidelity DNA Polymerase, KAPA HiFi™ HotStart ReadyMix or NEBNext® High-Fidelity 2X PCR Master Mix. Libraries were sequenced on an Illumina HiSeq® 2000. To correct for slight differences in the number of aligned reads from each library, 180 million reads were randomly extracted from each dataset, representing an average coverage of ~6X. The number of reads overlapping distinct low coverage regions of the human genome (Aird et.al. Genome Biology, 2011) are shown for each library. The NEBNext High-Fidelity 2X PCR Master Mix gives the most optimal performance of the three enzymes / master mixes tested.
    M0541
    Figure 2: Fidelity Comparisons of Different DNA Polymerases.
    Fidelity measurements of Taq DNA Polymerase (in Standard Taq Buffer), KAPA HiFi HotStart ReadyMix and NEBNext High-Fidelity 2X PCR Master Mix were measured side-by-side in a PCR-based mutation screening assay using a lacZ method modified from Kermekchiev et al., 2003. Values (n ≥ 2) are expressed relative to KAPA HiFi HotStart ReadyMix. The Q5 High-Fidelity Polymerase in the NEBNext High-Fidelity 2X PCR Master Mix has a level of fidelity >10X higher than that of the polymerase in the KAPA HiFi Hot Start ReadyMix.
    M0541
    Figure 3: Comparative Analysis of Different DNA Polymerases with Genomes of Varying % GC.
    Libraries of H. influenza, R. palustris or human genomic DNA were amplified using NEBNext High-Fidelity 2X PCR Master Mix, Phusion High-Fidelity PCR Master Mix with HF Buffer or KAPA HiFi HotStart PCR ReadyMix, and sequenced on an Illumina® HiSeq 2000. GC coverage plots were generated, with % GC content of 100 bp windows on the X axis. Normalized coverage is indicated by the blue circles, windows at GC% indicated by the red lines, and base quality at GC% indicated by the green line. NEBNext High-Fidelity 2X PCR Master Mix provides substantially reduced bias on all three genomic DNA samples.
    M0541
    Figure 4: Comparative Analysis of Different DNA Polymerases at Varying GC %s.
    Amplified libraries of human genomic DNA were generated using index primers and NEBNext High-Fidelity 2X PCR Master Mix, Phusion® High-Fidelity PCR Master Mix with HF Buffer or KAPA HiFi HotStart ReadyMix, and sequenced on an Illumina® HiSeq™ 2000. An equal number of reads from each dataset were randomly selected and the percentage of reads was plotted against GC content. NEBNext High-Fidelity 2X PCR Master Mix and KAPA HiF HotStart ReadyMix aligned closely to the expected read frequencies (shaded grey), while Phusion did not.

    Product Source

    An E. coli strain that carries the Q5 High-Fidelity DNA Polymerase gene.

    Reaction Volume Definition

    NEBNext High-Fidelity 2X PCR Master Mix, DNA template and 0.5 μM to 1.25 μM primers (depending on sample input) in a total reaction volume of 50 μl.

    Advantages and Features

    Applications

    • Next generation sequencing library construction
    • High fidelity PCR
    • Difficult amplification
    • High-throughput PCR

    Properties and Usage

    Storage Temperature

    -20°C

    Heat Inactivation

    No

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Non-Specific DNase Activity (16 hour):
      The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
    • PCR Amplification (737 base Human Genomic DNA, Master Mix):

      The polymerase master mix is tested in a polymerase chain reaction (PCR) using a control template and specific primers, resulting in the expected product.

    • Phosphatase Activity (PNPP):
      The product is tested in a reaction containing a p-nitrophenyl phosphate (PNPP), a chromogenic substrate for most phosphatases. After incubation there is no detectable phosphatase activity as determined by spectrophotometric analysis of released p-nitrophenol at 405 nm.

    Notes

    1. 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.

    References

    1. Anastassia Voronova Erin Coyne, Ashraf Al Madhoun, Joel V. Fair, Neven Bosiljcic, Catherine St-Louis, Grace Li, Sherry Thurig, Valerie A. Wallace, Nadine Wiper-Bergeron, and Ilona S. Skerjanc. (2013). Hedgehog Signaling Regulates MyoD Expression and Activity. J Biol Chem.. 288(6), 4389–4404. PubMedID: PMC3567689
    2. Lieve Naesens, Luke Guddat, Dianne Keough, André B.P. van Kuilenburg, Judith Meijer, Johan Vande Voorde and Jan Balzarini (2013). ROLE OF HUMAN HYPOXANTHINE GUANINE 
      PHOSPHORIBOSYLTRANSFERASE IN ACTIVATION 
      OF THE ANTIVIRAL AGENT T-705 (FAVIPIRAVIR). Molecular Pharmacology Fast Forward. 87247
    3. Hicham Bouabe, Klaus Okkenhaug (2013). A Protocol for Construction of Gene Targeting Vectors and Generation of Homologous Recombinant Embryonic Stem Cells. Methods in Molecular Biology. 1064, 337-354.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. What are the advantages of using NEBNext High-Fidelity 2X PCR Master Mix?
    2. Is NEBNext® High-Fidelity 2X PCR Master Mix the same as the Q5® High-Fidelity Master Mix?
    3. 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?
    4. How many cycles of PCR should I perform with NEBNext High-Fidelity 2X PCR Master Mix?
    5. Will NEBNext High-Fidelity 2X PCR Master Mix incorporate dUTPs?
    6. Are the DNA products produced NEBNext High-Fidelity 2X PCR Master Mix blunt-ended or do they have a single base 3’ overhang?
    1. PCR Using NEBNext® High-Fidelity 2X PCR Master Mix (M0541)