T4 Polynucleotide Kinase (3′ phosphatase minus) |NEB酶试剂 New England Biolabs

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

产品信息

T4 Polynucleotide Kinase catalyzes the transfer and exchange of Pi from the γ position of ATP to the 5´ -hydroxyl terminus of double- and single-stranded DNA and RNA, as well as nucleoside 3´-monophosphates (1-5). This modified version exhibits full kinase activity with no 3´ phosphatase activity (6,7).

产品来源

An E. coli strain that carries a plasmid encoding the modified T4 Polynucleotide Kinase gene.

产品类别:
Kinases Products,
RNA Modification

应用:
Phosphorylation (Kinase)

  • 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
    • M0236S     -20    
        T4 Polynucleotide Kinase (3′ phosphatase minus) M0236SVIAL -20 1 x 0.02 ml 10,000 units/ml
        T4 Polynucleotide Kinase Reaction Buffer B0201SVIAL -20 1 x 1 ml 10 X
    • M0236L     -20    
        T4 Polynucleotide Kinase (3′ phosphatase minus) M0236LVIAL -20 1 x 0.1 ml 10,000 units/ml
        T4 Polynucleotide Kinase Reaction Buffer B0201SVIAL -20 1 x 1 ml 10 X

  • 特性和用法

    单位定义

    One unit is defined as the amount of enzyme catalyzing the incorporation of 1 nmol of acid-insoluble [32P] in 30 minutes at 37°C (1). 

    Unit Assay Conditions: 
    1X T4 Polynucleotide Kinase Reaction Buffer, 66 µM [γ-32P] ATP (5 x 106 cpm/µmol), 0.26 mM 5´-hydroxyl-terminated salmon sperm DNA.

    反应条件

    1X T4 Polynucleotide Kinase Reaction Buffer
    Incubate at 37°C

    1X T4 Polynucleotide Kinase Reaction Buffer
    70 mM Tris-HCl
    10 mM MgCl2
    5 mM DTT
    (pH 7.6 @ 25°C)

    使用浓度

    10,000 units/ml

    贮存溶液

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

    热失活

    65°C for 20 minutes

    单位活性检测条件

    1X T4 Polynucleotide Kinase Reaction Buffer, 66 µM [γ-32P] ATP (5 x 106 cpm/µmol), 0.26 mM 5´-hydroxyl-terminated salmon sperm DNA.

  • 优势和特性

    Features

    5´ phosphorylation of DNA/RNA for subsequent ligation
    End-labeling of DNA or RNA (2)
    5´ phosphorylation of 3´ phosphorylated mononucleotides to generate a substrate (pNp) that can be added to the 3´ end of DNA or RNA by ligase activity (8)
    5´ end labeling of 3´ phosphorylated oligos (9)

  • 相关产品

    相关产品

    • t1010-monarch-plasmid-miniprep-kit
    • Monarch® DNA 胶回收试剂盒
    • Monarch® PCR & DNA 纯化试剂盒（5 μg）

    单独销售的组分

    • T4 多聚核苷酸激酶反应缓冲液

  • 注意事项

    1. Gaps can be phosphorylated with elevated levels of ATP. Nicks are not phosphorylated efficiently. CTP, GTP, TTP, UTP, dATP or dTTP can be substituted for ATP as a phosphate donor (1).
    2. Dephosphorylation prior to end-labeling can be avoided by utilizing the exchange reaction, although this results in lower specific activity labeling.
    3. Sufficient incorporation levels can be attained using the supplied buffer supplemented with 100 µM ADP in the final reaction. However, higher levels of incorporation with the exchange reaction are achieved when using the buffer described in (2).

  • 参考文献

    1. Richardson, CC. (1981). The Enzymes. 14, 299-314.
    2. Sambrook, J. et al. (1989). Molecular Cloning: A Laboratory Manual. 10.59-10.67,11.31-11.33.
    3. Cameron, V. and Uhlenbeck, O.C. (1977). Biochemistry . 16, 5120-5126.
    4. Berkner, K.L. and Folk, W.R. (1977). J. Biol. Chem.. 252, 3176-3184.
    5. Soltis, D.A. and Uhlenbeck, O.C (1982). J. Biol. Chem.. 257, 11332-11339.
    6. Wang, L.K. and Human, S (2001). J. Biol. Chem.. 276, 26868-26874.
    7. Wang, L.K. and Shuman, S. (2002). Nucl. Acids Res.. 30, 1073-1080.
    8. Vance, JR. and Wilson, T.E. (2001). Mol. Cell. Riot.. 21, 7191-7198.
    9. Interthal, H. et al. (2005). J. Biol. Chem.. 280, 36518-36528.

操作说明、说明书 & 用法

  • 操作说明

    1. Radioactive Labeling with T4 PNK or T4 PNK (3´ phosphatase minus)
    2. Non-radioactive Phosphorylation with T4 PNK or T4 PNK (3´ phosphatase minus)

  • 使用指南

    • Activity of DNA Modifying Enzymes in rCutSmart™ Buffer

FAQs & 问题解决指南

  • FAQs

    1. Will T4 PNK (3′ phosphate minus) work in rCutSmart Buffer?