Glycerol-Free High-Fidelity Pfu (HC)
PCR is used for the amplification of a next-generation sequencing (NGS) library. In addition to increasing the amount of DNA, the PCR amplification step is also used for DNA target enrichment for fragments that have an adaptor-ligated to each end. These will be the fragments that attach to the flow cell and will be sequenced, so it is important that this PCR amplification step does not introduce mutations to the NGS library, as a lower error rate means that less sequence coverage is required, saving expense and time.
Multiplex amplification
Glycerol-Free High-Fidelity Pfu (HC), was lyophilized with Lyo-Ready™ Pfu Reaction Buffer, 5x and stored at 37°C for 7 and 28 days. Lyophilization samples were tested in multiplexed PCR amplification of 7 targets (793 bp, 649 bp, 548 bp, 418 bp, 332 bp, 196 bp, 135 bp) against the same enzyme and reaction buffer without lyophilization (Wet Reference) to demonstrate stable after lyophilization allowing for extended storage periods and use in NGS library amplification.
Glycerol-Free High-Fidelity Pfu (HC), MDX203
Available in 0.025 mL (500 Units) or 1 mL (20,000 Units) aliquots
Description
High-Fidelity Pfu is a thermostable high fidelity DNA polymerase enzyme, isolated from Pyrococcus furiosus. The Pfu polymerase enzyme replicates DNA in the 5´→3´ direction in the presence of magnesium, but also possesses 3´→5´ exonuclease (proofreading) activity. Base misincorporations that may occur during polymerization are rapidly excised by this proofreading activity. High-Fidelity has an error rate of 3.0 x 10-6 generating blunt-ended amplicons up to 5 kb in length. Glycerol-Free High-Fidelity Pfu (HC) is supplied in a glycerol-free storage buffer and is accompanied by a 5x Reaction Buffer that contains magnesium, dNTPs and excipients required for lyophilization.
Specifications
| Description | High concentration, high-fidelity Taq DNA polymerase with separate Pfu Polymerase Dilution Buffer and10x Pfu Reaction Buffer. |
| Concentration | 20 U/µL |
| Appearance | Clear, colorless solution |
| Application | PCR, NGS library preparation, cloning, protein expression |
| Sample type | cDNA, DNA |
| Presentation | 3 vials |
| Storage | -20 °C |
| Mix stability | See outer label |
| Consistency | ± 0.5 Ct variance between test and reference sample |
| DNA Contamination | None detected in PCR amplification with traces overlay with the negative control on E. coli and mouse genomic DNA specific targets. |
| DNase Contamination | No detectable degradation |
产品资料
分子诊断的原料试剂解决方案分子诊断的原料试剂解决方案
Pfu高保真热启动聚合酶Pfu高保真热启动聚合酶
二代测序 (NGS)二代测序 (NGS)
A Comprehensive Guide to Essential Reagents for High-Throughput Molecular Diagnostic Assays (MDx)Accelerating Efficiency: A Comprehensive Guide to Essential Reagents for High-Throughput Molecular Diagnostic Assays (MDx)
FAQs: High-Fidelity Pfu
在自然界中，DNA聚合酶在DNA链延长过程中纠错核苷酸整合的能力对于个体生存至关重要。这被称为“校对活动”，发生在3′到5′方向。聚合酶的这种活性还能去除3'端（A-端）的未配对核苷酸，产生钝端。
If possible, we would recommend an additional clean-up of affected samples, alternatively High-Specificity Pfu HS Mix (MDX006) would be recommended, as it is designed to work in the presence of inhibitors.
High-Fidelity Pfu has been validated for templates up to 5 kb.
DNA target enrichment is a pre-sequencing DNA preparation step where DNA sequences are either directly amplified (multiplex PCR-based) or captured (hybrid capture-based). These enriched DNA fragments can then be sequenced using DNA sequencers.
A lyophilized PCR molecular test will remain stable for up to 2 years at ambient temperature if correctly stored in sealed pouches.
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