RNase HII |NEB酶试剂 New England Biolabs

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产品信息

Ribonuclease HII (RNase HII) is an endoribonuclease that preferentially nicks 5´ to a ribonucleotide within the context of a DNA duplex. The enzyme leaves 5´ phosphate and 3´ hydroxyl ends (1). RNase HII will also nick at multiple sites along the RNA portion of an Okazaki fragment.

产品来源

An E. coli strain containing a genetic fusion of the RNase HII gene (rnhB) from E. coli and the gene coding for maltose binding protein (MBP). Following affinity chromatography, RNase HII is cleaved from the fusion construct by Factor Xa and then purified away from both MBP and Factor Xa. RNase HII cleaved from MBP has four additional amino acids at its N-terminus (Ile-Ser-Glu-Phe).

产品类别:
RNases

  • 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
    • M0288S     -20    
        RNase HII M0288SVIAL -20 1 x 0.05 ml 5,000 units/ml
        ThermoPol® Reaction Buffer B9004SVIAL -20 1 x 1.5 ml 10 X
    • M0288L     -20    
        RNase HII M0288LVIAL -20 1 x 0.25 ml 5,000 units/ml
        ThermoPol® Reaction Buffer B9004SVIAL -20 1 x 1.5 ml 10 X

  • 特性和用法

    单位定义

    One unit is defined as the amount of enzyme required to yield a fluorescence signal consistent with the nicking of 100 picomol of synthetic double-stranded DNA substrate containing a single ribonucleotide near the quencher of a fluorophore/quencher pair in 30 minutes at 37°C in 1X ThermoPol Buffer.

    反应条件

    1X ThermoPol® Reaction Buffer
    Incubate at 37°C

    1X ThermoPol® Reaction Buffer
    20 mM Tris-HCl
    10 mM (NH4)2SO4
    10 mM KCl
    2 mM MgSO4
    0.1% Triton® X-100
    (pH 8.8 @ 25°C)

    贮存溶液

    20 mM Tris-HCl
    100 mM NaCl
    1 mM DTT
    1 mM EDTA
    50% Glycerol

    热失活

    否

    单位活性检测条件

    1X ThermoPol Reaction Buffer with 30 nM of a synthetic dsDNA 26-mer containing an internal ribonucleotide in a total reaction volume of 150 µl.

  • 优势和特性

    应用特性

    Nicking of products generated with a polymerase that will incorporate ribonucleotides
    Generation of a double-stranded break at the site of an incorporated ribonucleotide when used with T7 Endo I
    Degradation of the RNA portion of Okazaki fragments

  • 相关产品

    单独销售的组分

    • ThermoPol® 反应缓冲液套装

  • 注意事项

    1. RNase HII displays reduced hybridase activity compared to RNase H (2).
    2. Okazaki fragments are preferentially nicked 5´ to the ribonucleotide at the junction of the RNA/DNA sequence (1).
    3. RNase HII prefers a dsDNA duplex containing a single ribonucleotide over a RNA/DNA hybrid substrate (1) or an Okazaki fragment (3).
    4. Incubation with 0.1% SDS is sufficient to inactivate RNase HII.

  • 参考文献

    1. Rydberg, B. and Game, J. (2002). Proc. Natl. Acad. Sci.. 99,
    2. Itaya, M. (1990). Proc. Natl. Acad. Sci.. 87,
    3. Nichols, N.M. Unpublished Observation.

操作说明、说明书 & 用法

  • 操作说明

    1. Nicking a single ribonucleotide site within a dsDNA substrate or an Okazaki fragment with RNase HII (NEB #M0288)

工具 & 资源

  • 选择指南

    • Activities of DNA Repair Enzymes and Structure-specific Endonucleases
    • Properties of DNA Repair Enzymes and Structure-specific Endonucleases

FAQs & 问题解决指南

  • FAQs

    1. RNase HII cannot be heat-inactivated. How can RNase HII be inactivated?
    2. Which side of the ribonucleotide does RNase HII cut?