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T4 DNA Ligase Ⅱ STAR

DNA Ligase、Polydeoxyribonucleotide synthase [ATP]

价格 ￥200.00 （ 供应商现货 ： 3-5个工作日）
货号 UA070048
规格
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产品介绍

产品介绍 评论(0)

产品规格

  • 分子别名

    DNA Ligase、Polydeoxyribonucleotide synthase [ATP]

  • 表达宿主

    E.coli

  • 分子量

    81 kD

  • 纯度

    ＞95% by SDS-PAGE & SEC-HPLC

  • 活性

    400U/μl

  • 标记

    Unconjugated

  • 性状

    Liquid

  • 缓冲体系

    10 mM Tris-HCl、50 mM KCl、1 mM DTT、0.1 mM EDTA、50% Glycerol （pH 7.4 @ 25°C）

  • 储存条件

    Store at -25 ~ -15℃ for 1 years

  • 文献引用

    [1] Williamson A, Pedersen H. Recombinant expression and purification of an ATP-dependent DNA ligase from Aliivibrio salmonicida. Protein Expr Purif. 2014 May; 97:29-36.
    [2] Liu X, Huang A, Luo D, Liu H, Han H, Xu Y, Liang P. Use of adenylate kinase as a solubility tag for high level expression of T4 DNA ligase in Escherichia coli. Protein Expr Purif. 2015 May;109:79-84.

背景介绍

  • T4 DNA ligase is a type of DNA ligase. DNA ligase catalyzes the formation of phosphodiester bonds at single-stranded DNA breaks in double-stranded DNA in vivo. DNA ligase has important biological functions in organisms. In DNA repair and recombination, DNA ligase plays a role in connecting gaps. In the process of DNA replication, the synthesis of the lagging strand is discontinuous, and DNA ligase connects the discontinuous DNA strand into a continuous DNA strand. T4 DNA Ligase Ⅱ is a recombinant protein fusion of T4 DNA Ligase and adenylate kinase, The purified T4 DNA Ligase Ⅱ not only is fully active for DNA ligation, but also can use ADP in addition to ATP as energy source since adenylate kinase converts ADP to ATP and AMP. It does not contain DNA endonuclease, exonuclease and phosphatase, and does not contain RNA enzyme.

产品组分

  • Storage Solution: 400U/μl T4 DNA Ligase、10 mM Tris-HCl、50 mM KCl、1 mM DTT、0.1 mM EDTA、50% Glycerol (pH 7.4 @ 25°C) 10*Reaction Buffer: 500 mM Tris-HCl、100 mM MgCl2、10 mM ATP、100 mM DTT (pH 7.5 @ 25°C)

操作步骤

  • 1. Set up the following reaction in a microcentrifuge tube on ice. Add the following components in sequence. Note that the table shows a ligation using a molar ratio of 1:5 vector to insert for the indicated DNA sizes.

    Components

    Volume 20μl

    10* T4 DNA Ligase Buffer

    2 μl

    Vector DNA (such as pET-28a 5369bp)

    50ng

    Insert DNA (530bp)

    25ng

    Nuclease-free water

    Up to 20 μL

    T4 DNA Ligase Ⅱ

    1μl

    2. Gently mix the reaction through the up and down pipette, and inhale the liquid briefly.

    3. For sticky ends, incubate at 16°C overnight or at room temperature for 10 minutes.

    4. For blunt ends or single base overhangs, incubate overnight at 16°C or room temperature for 2 hours (alternatively, high concentrations of T4DNA ligase can be used for 10 minutes of ligations).

    5. Chill on ice and transform 1-5 μl of the reaction into competent cells.

注意事项

  • 1.ATP is an essential cofactor in the reaction. This is in contrast to E. coli DNA Ligase, which requires NAD as a cofactor.

    2.If T4 DNA Ligase is to be diluted, it is recommended that it be diluted with 50% glycerol in storage buffer and stored at -20°C. 3. Room temperature ligation

酶活定义
  • One unit is the amount of enzyme required to ligate 50% of the HindIII-digested λ DNA fragments [DNA 5´-end concentration of 0.12 µM (300 μg/ml)] within 30 minutes at 16°C in a 20 µl reaction system and in 1X T4 DNA Ligase Reaction Buffer.

  • 生物活性

    • The two DNA strands after enzymatic digestion and purification were used as substrate for enzyme linkage and incubated at 25℃ for 2 hours. The reaction was analyzed by 1% agarose gel electrophoresis.
      M: DNA marker
      Lane 1 negative control
      Lane 2 Experimental result

  • 电泳

    • 2μg (R: reducing condition, N: non-reducing condition).

  • 体积排阻色谱(SEC-HPLC)

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