GpC Methyltransferase (M.CviPI) |NEB酶试剂 New England Biolabs

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产品信息

产品来源

The GpC Methyltransferase, M.CviPI, is isolated from a strain of E. coli which contains the methyltransferase gene from Chlorella virus. This construct is fused to the maltose binding protein (MBP).

产品类别:
Methyltransferases for Epigenetics Products,
Methyltransferases Products

  • 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
    • M0227S     -20    
        GpC Methyltransferase (M.CviPI) M0227SVIAL -20 1 x 0.05 ml 4,000 units/ml
        GC Reaction Buffer B0227SVIAL -20 1 x 1.5 ml 10 X
        S-adenosylmethionine (SAM) B9003SVIAL -20 1 x 0.1 ml 32 mM
    • M0227L     -20    
        GpC Methyltransferase (M.CviPI) M0227LVIAL -20 1 x 0.25 ml 4,000 units/ml
        GC Reaction Buffer B0227SVIAL -20 1 x 1.5 ml 10 X
        S-adenosylmethionine (SAM) B9003SVIAL -20 1 x 0.1 ml 32 mM

  • 特性和用法

    单位定义

    One unit is defined as the amount of enzyme required to protect 1 µg of λ DNA in a total reaction volume of 20 µl in 1 hour at 37°C against cleavage by HaeIII restriction endonuclease. 

    反应条件

    1X GC Reaction Buffer
    Supplement with 160 µM S-腺苷甲硫氨酸（SAM）
    Incubate at 37°C

    1X GC Reaction Buffer
    50 mM NaCl
    50 mM Tris-HCl
    10 mM DTT
    (pH 8.5 @ 25°C)

    对照保护实验

    M.CviPI is incubated with 1 µg λ DNA in 20 µl 1X GC Reaction Buffer and 160 µM S-adenosylmethionine, for one hour at 37°C. The extent of protection by M.CviPI is determined by the addition of 30 µl NEBuffer 2 containing 10 units of HaeIII restriction endonuclease. Incubation for 1 hour at 37°C is followed by analysis on an agarose gel.

    贮存溶液

    15 mM Tris-HCl
    0.2 M NaCl
    1 mM DTT
    0.1 mM EDTA
    200 µg/ml BSA
    50% Glycerol
    pH 7.4 @ 25°C

    热失活

    65°C for 20 minutes

  • 优势和特性

    应用特性

    • Blocking restriction endonuclease cleavage
    • Altering the physical properties of DNA
    • Uniform [3H]-labeling of DNA

  • 相关产品

    单独销售的组分

    • S-腺苷甲硫氨酸（SAM）

  • 注意事项

      • S-腺苷甲硫氨酸（SAM）以 32 mM 浓度溶解于 0.005 M 硫酸和 10% 乙醇溶液中提供。这些条件下，SAM 可在 -20℃ 稳定储存长达六个月的时间。
      • SAM 在 37℃，pH 7.5 的条件下不稳定；因此，当反应温育时间超过 4 小时，需补充 SAM。
    2. 需要新鲜配置的 DTT 来实现最佳活性。为获得最佳结果，每次使用前新配制检测缓冲液。
    3. 胞嘧啶残基的甲基化影响 DNA 的物理特性，包括降低 Z-DNA 形成的自由能（1）、增加 DNA 的螺旋程度（2）、以及改变十字形突出的动力学特性（3）。在化学测序过程（4），由于对联氨的反应活性降低，5-甲基胞嘧啶的位置可被识别。
    4. MgCl2 is not required as a cofactor.

  • 参考文献

    1. Zacharias, W. et al. (1988). Biochemistry. 27, 2970-2978.
    2. Gruenbaum, Y. et al. (1982). Nature. 295, 620-621.
    3. Murchie, A.I. and Lilley, D.M. (1989). J. Mol. Biol.. 205, 593-602.
    4. Ohmori, H. et al. (1978). Nucl. Acids. Res. 5, 1479-1485.
    5. Xu, S. et al. (1998). Nucl. Acids Res.. 26, 3961-3966.
    6. Kladde, M.P. et al. (1991). Methods Enzymol. . 304, 431-447.

操作说明、说明书 & 用法

  • 操作说明

    1. Recommended Protocol for 3H labeling of DNA
    2. Recommended Protocol for Methylation of Genomic DNA

  • 使用指南

    • Activity of DNA Modifying Enzymes in rCutSmart™ Buffer

FAQs & 问题解决指南

  • FAQs

    1. Will GpC Methyltransferase (M. CviPI) work in rCutSmart buffer?
    2. Will any NEB methyltransferases (methylases) work on RNA?

  • 实验技巧

    Make sure the reaction buffer is diluted out just before doing the methylation reaction. This enzyme requires fresh DTT for optimal activity.