DNA Polymerase I, Large (Klenow) Fragment | NEB酶试剂 New England Biolabs

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DNA Polymerase I, Large (Klenow) Fragment

DNA Polymerase I, Large (Klenow) fragment was originally derived as a proteolytic product of E.coli DNA polymerase that retains polymerase and 3’ —> 5’ exonuclease activity

  • Removal of 3’ overhangs or fill-in of 5’ overhangs to form blunt ends
  • Lacks 5’ —> 3’ exonuclease activity
  • Generates probes using random primers
  • Second strand cDNA synthesis

精选视频

DNA Polymerase I, Large (Klenow) Fragment | NEB酶试剂 New England Biolabs

DNA Blunting Tutorial

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库存
货号 浓度 规格 目录价 北京 上海 广州 成都 苏州
M0210V 5,000 units/ml 100 units ¥359.00 有 有 无 无 有
M0210S 5,000 units/ml 200 units ¥679.00 有 有 有 有 有
M0210L 5,000 units/ml 1,000 units ¥2,699.00 有 有 有 无 有
M0210M 50,000 units/ml 1,000 units ¥2,699.00 有 有 有 无 无
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产品信息

DNA Polymerase I, Large (Klenow) Fragment is a proteolytic product of E. coli DNA Polymerase I which retains polymerization and 3’→ 5′ exonuclease activity, but has lost 5’→ 3′ exonuclease activity (1). Klenow retains the polymerization fidelity of the holoenzyme without degrading 5′ termini.

重点

 

产品来源

An E. coli strain that contains the E. coli polA gene that has had its 5’→3′ exonuclease domain removed.

产品类别:
DNA Manipulation Products

应用:
Blunting,
DNA Sequencing,
Polymerases for DNA Manipulation,

RT-PCR & cDNA Synthesis,
RT-qPCR, RT-PCR and cDNA Synthesis,

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
    • M0210V     -20    
        DNA Polymerase I, Large (Klenow) Fragment M0210VVIAL -20 1 x 0.02 ml 5,000 units/ml
        NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X
    • M0210S     -20    
        DNA Polymerase I, Large (Klenow) Fragment M0210SVIAL -20 1 x 0.04 ml 5,000 units/ml
        NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X
    • M0210L     -20    
        DNA Polymerase I, Large (Klenow) Fragment M0210LVIAL -20 1 x 0.2 ml 5,000 units/ml
        NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X
    • M0210M     -20    
        DNA Polymerase I, Large (Klenow) Fragment M0210MVIAL -20 1 x 0.02 ml 50,000 units/ml
        NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 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 37°C.

    反应条件

    1X NEBuffer™ 2
    Incubate at 25°C

    1X NEBuffer™ 2
    50 mM NaCl
    10 mM Tris-HCl
    10 mM MgCl2
    1 mM DTT
    (pH 7.9 @ 25°C)

    贮存溶液

    25 mM Tris-HCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    pH 7.4 @ 25°C

    热失活

    75°C for 20 min

    分子量

    理论上的: 68000 daltons

    5′ – 3′ 核酸外切酶

    No

    3′ – 5′ 核酸外切酶

    Yes

    链置换

    +

    单位活性检测条件

    1X NEBuffer 2, 33 μM dNTPs including [3H]-dTTP and 70 μg/ml denatured herring sperm DNA.

    错配率

    ~ 18×10-6bases

  • 优势和特性

    应用特性

    • DNA sequencing by the Sanger dideoxy method (2)
    • Fill-in of 5´ overhangs to form blunt ends (3)
    • Removal of 3´ overhangs to form blunt ends (3)
    • Second strand cDNA synthesis
    • Second strand synthesis in mutagenesis protocols (4)

  • 相关产品

    相关产品

    • Deoxynucleotide (dNTP) Solution Set
    • Deoxynucleotide (dNTP) Solution Mix
    • Monarch® Plasmid Miniprep Kit 
    • Monarch® DNA Gel Extraction Kit
    • Monarch® PCR & DNA Cleanup Kit (5 μg)

    单独销售的组分

    • NEBuffer 2

  • 注意事项
    1. Protocol for blunting ends by 3′ overhang removal and 3′ recessed end fill-in: 
      • DNA should be dissolved in 1X NEBuffer 1-4 or T4 DNA Ligase Reaction Buffer supplemented with 33 μM each dNTP. Add 1 unit DNA Polymerase I, Large (Klenow) Fragment per microgram DNA and incubate 15 minutes at 25°C. Stop reaction by adding EDTA to a final concentration of 10 mM and heating at 75°C for 20 minutes. 
      • CAUTION: Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times may result in recessed ends due to the 3’→ 5′ exonuclease activity of the enzyme.
    2. When DNA Polymerase I, Large (Klenow) Fragment is used to sequence DNA using the dideoxy method of Sanger et al., 1 unit/5 μl reaction volume is recommended.
    3. DNA Polymerase I, Large (Klenow) Fragment is also active in all four NEBuffers and T4 DNA Ligase Reaction Buffer when supplemented with dNTPs.

  • 参考文献
    1. Jacobsen, H., Klenow, H. and Overgaard-Hansen, K. (1974). Eur. J. Biochem.. 45, 623-627.
    2. Sanger, F. et al. (1977). Proc. Natl. Acad. Sci. USA. 74, 5463-5467.
    3. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (2nd ed.), 5.40-5.43. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
    4. Gubler, U. (1987). In S.L. Berger and A.R. Kimmel(Ed.), Methods in Enzymology. 152, 330-335. San Diego: Academic Press.
    5. Bebenek, K., Joyce, C.M., Fitzgerald, M.P. and Kunkel, T.A. (1990). J. Bio. Chem . 265, 13878-13887.

操作说明、说明书 & 用法

  • 操作说明
    1. Protocol for blunting ends by 3′ overhang removal and fill-in of 3′ recessed (5′ overhang) ends using DNA Polymerase I, Large (Klenow) Fragment (M0210)

  • 使用指南
    • Activity of DNA Modifying Enzymes in rCutSmart™ Buffer

工具 & 资源

  • 选择指南
    • Blunting Selection Chart
    • DNA Polymerase Selection Chart

  • Web 工具
    • NEBcloner®

FAQs & 问题解决指南

  • FAQs
    1. Can DNA Polymerase I, Large (Klenow) Fragment be used in other NEBuffers, including rCutSmart?
    2. Can DNA Polymerase I, Large (Klenow) Fragment be used to blunt DNA?
    3. Can DNA Polymerase I, Large (Klenow) Fragment be used to fill in 3′ overhangs?
    4. Can DNA Polymerase I, Large (Klenow) Fragment be used to chew back 5′ overhangs?
    5. Can DNA Polymerase I, Large (Klenow) Fragment be heat inactivated?
    6. Are the nucleotides needed to remove a 3′ overhang with DNA Polymerase I, Large (Klenow) Fragment?
    7. What are the main causes for blunting reaction failure using DNA Polymerase I, Large (Klenow) Fragment?
    8. Can DNA Polymerase I, Large (Klenow) Fragment be used in labeling reactions and partial fill in reactions?
    9. Is DNA Polymerase I, Large (Klenow) Fragment the enzyme of choice for chewing back 3′ overhangs and filling in 5′ overhangs (3′ recessed ends)?
    10. Are NEB DNA Polymerases supplied with dNTPs?

  • 实验技巧
    Excess enzyme, temperatures above 25°C, or limited dNTP concentrations can cause excessive 3’ ? 5’ exonuclease degradation, which eliminates blunt end formation.

引用 & 技术文献

  • 引用文献

    产品引用文献查找工具

    DNA Polymerase I, Large (Klenow) Fragment | NEB酶试剂 New England Biolabs Powered by Bioz See more details on Bioz

质控、安全 & 法规

  • 质控分析
    每一新批次的 NEB 产品都要经过质控，以满足指定的质量标准，对特定产品的质量标准和每一批次的检测数据可以通过产品说明表格、COA、产品信息卡或者产品手册进行查阅或下载。关于 NEB 产品质控的详细信息，可从此处查阅。

  • 产品说明与变更通知

    产品说明与变更通知

    产品说明表包含产品的储存温度、有效期和规格，这些文件的命名规则如下：[货号]_[规格]_[版本]

    • M0210M_v1
    • M0210S_L_v1

  • CoA
    CoA 文件包含单个批次产品的储存温度、有效期和质控，这些文件的命名规则如下： [货号]_[规格]_[版本]_[Lot#]

    • M0210M_v1_0891509
    • M0210S_L_v1_0891509
    • M0210S_L_v1_0891512
    • M0210S_L_v1_0891603
    • M0210M_v1_0891603
    • M0210M_v1_0891608
    • M0210S_L_v1_0891608
    • M0210M_v1_0891703
    • M0210S_L_v1_0891703
    • M0210M_v1_0891709
    • M0210S_L_v1_0891709
    • M0210M_v1_0891803
    • M0210S_L_v1_0891803
    • M0210L_v1_10007540
    • M0210L_v1_10025743
    • M0210S_v1_10024368
    • M0210L_v1_10028508
    • M0210M_v1_10028233
    • M0210S_v1_10028423
    • M0210L_v1_10034214
    • M0210M_v1_10034564
    • M0210S_v1_10034001
    • M0210L_v1_10036837
    • M0210S_v1_10037080
    • M0210L_v0_10041498
    • M0210L_v1_10046211
    • M0210S_v1_10043965
    • M0210S_v1_10050337
    • M0210L_v1_10053482
    • M0210S_v1_10055962
    • M0210L_v1_10057607
    • M0210L_v1_10058269
    • M0210S_v1_10058172
    • M0210L_v1_10062077
    • M0210M_v1_10057608
    • M0210S_v1_10062081
    • M0210L_v1_10065299
    • M0210S_v1_10066373
    • M0210L_v1_10075466
    • M0210S_v1_10075464
    • M0210S_v1_10079480
    • M0210M_v1_10087408
    • M0210L_v1_10088146
    • M0210S_v1_10087380
    • M0210S_v1_10094981
    • M0210L_v1_10100634
    • M0210M_v1_10101977
    • M0210L_v1_10106852
    • M0210S_v1_10107645
    • M0210S_v1_10113999
    • M0210L_v1_10117205
    • M0210M_v1_10117349
    • M0210M_v1_10123077
    • M0210S_v1_10127909
    • M0210L_v1_10136292
    • M0210M_v1_10137266
    • M0210L_v1_10149552
    • M0210S_v1_10150626
    • M0210L_v1_10155130
    • M0210M_v1_10154966
    • M0210L_v1_10162372

  • SDS
    以下 SDS 文件可以帮助您安全地使用该产品

    • DNA Polymerase I, Large (Klenow) Fragment
    • NEBuffer™ 2