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NEBNext® High-Fidelity 2X PCR Master Mix
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.
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产品信息
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.
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 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. 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.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.
产品来源
An E. coli strain that carries the Q5 High-Fidelity DNA Polymerase gene.
产品类别:
Q5® High-Fidelity DNA Polymerases Products
应用:
Polymerases for NGS Library Preparation,
Fast PCR,
High-Fidelity PCR,
DNA Library Preparation,
DNA Amplification, PCR & qPCR,
Specialty PCR,
Routine PCR,
PCR
产品组分信息
产品组分信息
本产品提供以下试剂或组分：
NEB #
名称
组分货号
储存温度
数量
浓度
M0541S
-20
NEBNext® High-Fidelity 2X PCR Master Mix
M0541SVIAL
-20
1 x 1.25 ml
2 X
M0541L
-20
NEBNext® High-Fidelity 2X PCR Master Mix
M0541SVIAL
-20
5 x 1.25 ml
2 X
特性和用法
热失活
否
优势和特性
应用特性
Next generation sequencing library amplification
High fidelity PCR
Difficult amplification
High-throughput PCR
相关产品
相关产品
NEBNext® End Repair Module
NEBNext® dA-Tailing Module
NEBNext® Quick Ligation Module
NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1)
NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 2)
NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1)
NEBNext Multiplex Oligos for Illumina (Index Primers Set 3)
NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 4)
NEBNext® Multiplex Oligos for Illumina® (96 Index Primers)
注意事项
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.
操作说明、说明书 & 用法
操作说明
PCR Using NEBNext® High-Fidelity 2X PCR Master Mix (M0541)
工具 & 资源
Web 工具
Tm Calculator
FAQs & 问题解决指南
FAQs
What are the advantages of using NEBNext High-Fidelity 2X PCR Master Mix?
Is NEBNext® High-Fidelity 2X PCR Master Mix the same as the Q5® High-Fidelity Master Mix?
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?
How many cycles of PCR should I perform with NEBNext High-Fidelity 2X PCR Master Mix?
Will NEBNext High-Fidelity 2X PCR Master Mix incorporate dUTPs?
Are the DNA products produced NEBNext High-Fidelity 2X PCR Master Mix blunt-ended or do they have a single base 3’ overhang?
问题解决指南
PCR Troubleshooting Guide
引用 & 技术文献
引用文献
NEBNext Product Citations Search Tool
Use this tool to find citations related to NEBNext products. Search by product, keyword or instrument name – use the filters to select specific product areas.
更多引用文献
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
Schmid-Burgk, J.L. et al. () LAMP-Seq: Population-scale COVID-19 diagnostics using a compressed barcode space bioRxiv; DOI: https://doi.org/10.1101/2020.04.06.025635
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