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商品編號


M0541S-250 reactions



品牌


紐英倫(NEB)



公司


New England Biolabs



公司分類(lèi)


PCR, qPCR & Amplification Technologies




250 reactions


Concentration

2X






商品信息

Description:
?
The
NEB
Next 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
Thermo
stable 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.
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
NEB
Next? 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
BIOLOG
y, 2011) are shown for each library. The
NEB
Next 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
NEB
Next 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
NEB
Next 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
NEB
Next 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.
NEB
Next 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
NEB
Next 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.
NEB
Next 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
NEB
Next 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.
Notes:
To ensure optimal performance, the master mix should be thawed and res
USP
ended prior to use. St
ABI
lity testing using up to 20 freeze/thaw cycles has shown no negative effect on master mix performance. The
NEB
Next High-Fidelity 2X PCR Master Mix may be liquid at -20°C.