Phusion® High-Fidelity DNA Polymerase |NEB酶试剂 New England Biolabs

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产品信息

High-Fidelity DNA Polymerases are important for applications in which the DNA sequence needs to be correct after amplification. Its unique structure, a novel Pyrococcus-like enzyme fused with a processivity-enhancing domain, increases fidelity and speed. Phusion DNA Polymerase is an ideal choice for cloning and can be used for long or difficult amplicons. With an error rate > 50-fold lower than that of Taq DNA Polymerase and 6-fold lower than that of Pyrococcus furiosus DNA Polymerase (1), Phusion is one of the most accurate thermostable polymerases available. Phusion DNA Polymerase possesses 5´→ 3´ polymerase activity, 3´→ 5´ exonuclease activity and will generate blunt-ended products.

Phusion DNA Polymerase is supplied with standard 5X Phusion HF Buffer, as well as 5X Phusion GC Buffer, which can be used for complex or GC-rich templates. Each of these buffers contains MgCl2 (1.5 mM at the final [1X] reaction concentration). Reactions can also be optimized using the provided DMSO or MgCl2 solutions.

Phusion® High-Fidelity DNA Polymerase |
Fidelity assays were performed using a lacI-based method modified from Frey & Suppman, 1995.
Phusion® High-Fidelity DNA Polymerase |
A 3.8 kb fragment was amplified from 50 ng of Jurkat gDNA using different polymerases. Reactions were carried out according to the manufacturer’s recommended conditions. Extension times are indicated (in minutes). Ladder L is a 1 kb DNA Ladder (NEB# N3232).

产品来源

An E. coli strain that carries the Phusion DNA Polymerase gene.

产品类别:
Phusion® High-Fidelity DNA Polymerases Products

应用:
Gibson Assembly®,
Long Range PCR,
Fast PCR,

High-Fidelity PCR,
Multiplex PCR,
Specialty PCR,
Routine PCR,

PCR

  • 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
    • M0530S     -20    
        Phusion® High-Fidelity DNA Polymerase M0530SVIAL -20 1 x 0.05 ml 2,000 units/ml
        MgCl2 solution B0510AVIAL -20 1 x 1.5 ml 50 mM
        DMSO B0515AVIAL -20 1 x 0.5 ml 100 %
        Phusion® HF Buffer Pack B0518SVIAL -20 2 x 1.5 ml 5 X
        Phusion® GC Buffer Pack B0519SVIAL -20 1 x 1.5 ml 5 X
    • M0530L     -20    
        Phusion® High-Fidelity DNA Polymerase M0530LVIAL -20 1 x 0.25 ml 2,000 units/ml
        MgCl2 solution B0510AVIAL -20 2 x 1.5 ml 50 mM
        DMSO B0515AVIAL -20 1 x 0.5 ml 100 %
        Phusion® HF Buffer Pack B0518SVIAL -20 6 x 1.5 ml 5 X
        Phusion® GC Buffer Pack B0519SVIAL -20 2 x 1.5 ml 5 X

  • 特性和用法

    单位定义

    One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 74°C.

    贮存溶液

    20 mM Tris-HCl
    100 mM KCl
    1 mM DTT
    0.1 mM EDTA
    200 µg/ml BSA
    50% Glycerol
    1X stabilizers
    pH 7.4 @ 25°C

    热失活

    否

    5′ – 3′ 核酸外切酶

    No

    3′ – 5′ 核酸外切酶

    Yes

    单位活性检测条件

    25 mM TAPS-HCl (pH 9.3 @ 25°C), 50 mM KCl, 2 mM MgCl2, 1 mM b-mercaptoethanol, 200 µM dNTPs including [3H]-dTTP and 15 nM primed M13 DNA.

  • 优势和特性

    Features

    • Robust Reactions – Maximal success with minimal optimization
    • Extreme Fidelity – >50X greater than Taq.
    • High Speed – Dramatically reduced extension times (10X faster than Pfu)
    • High Yield – Increased product yield using minimal amount of enzyme
    • Versatile – Can be used for routine PCR as well as long or difficult templates

    应用特性

    • PCR
    • Cloning
    • Long or Difficult Amplification
    • High-Throughput PCR

  • 相关产品

    相关产品

    • dNTP 套装
    • dNTP 混合液
    • MgCl2 溶液
    • Phusion® High-Fidelity PCR Kit
    • Phusion® 超保真 PCR 预混液（提供 HF 缓冲液）
    • Phusion® 超保真 PCR 预混液（提供 GC 缓冲液）
    • dNTP 混合液
    • Phusion® HF 反应缓冲液套装
    • Phusion® GC 反应缓冲液套装

  • 参考文献

    1. Frey, B. and Suppman, B. (1995). BioChemica. 2, 34-35.
    2. Chester, N. and Marshak, D.R. (1993). Analytical Biochemistry. 209, 284-290.

操作说明、说明书 & 用法

  • 操作说明

    1. PCR Protocol for Phusion® High-Fidelity DNA Polymerase (M0530)

  • 使用指南

    • Activity of Restriction Enzymes in PCR Buffers
    • Guidelines for PCR Optimization with Thermophilic DNA Polymerases

工具 & 资源

  • 选择指南

    • DNA Polymerase Selection Chart
    • Thermophilic DNA Polymerases

  • Web 工具

    • Tm Calculator

FAQs & 问题解决指南

  • FAQs

    1. What are the differences between the various available Phusion® products?
    2. My results are not as expected. Where can I find troubleshooting help?
    3. What ends will my PCR products have?
    4. What does exonuclease activity mean for a DNA polymerase?
    5. Does Phusion® High-Fidelity DNA Polymerase exhibit a strand displacement activity?
    6. What are the properties of this polymerase (fidelity, product ends, max amplicon, modified base incorporation, etc.)?
    7. How should I determine the appropriate annealing temperature for my reaction?
    8. What should the final primer concentration be in my PCR?
    9. My template is GC-rich or supercoiled. How can I optimize my product yield using Phusion® High-Fidelity DNA Polymerase?

  • 问题解决指南

    • PCR Troubleshooting Guide

  • 实验技巧

    Phusion has different annealing temperature requirements than most PCR enzymes. Please check out NEB’s Tm Calculator to help you determine your optimal Phusion annealing temperature.