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.
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.
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.
Figure 2: Fidelity comparisons of different DNA polymerasesFidelity 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.
Figure 3: Comparative analysis of different DNA polymerases with genomes of varying percent 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.
Figure 4: Comparative analysis of different DNA polymerases at varying percent GC
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.
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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.
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
Licenses
This product is licensed for research and commercial use from Bio-Rad Laboratories, Inc., under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645, and corresponding patents in other countries. No rights are granted for use of the product for Digital PCR or real-time PCR applications, with the exception of quantification in Next Generation Sequencing workflows.
For additional information or to inquire about commercial use, please contact busdev@neb.com.
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