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DNA Polymerase I Large (Klenow) Fragment

Klenow Fragment、DNA Polymerase I Large (Klenow) Fragment

价格 ￥550.00 （ 供应商现货 ： 3-5个工作日）
货号 UA070056
规格
数量
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产品介绍

产品介绍 评论(0)

产品规格

  • 分子别名

    Klenow Fragment、DNA Polymerase I Large (Klenow) Fragment

  • 表达宿主

    E.coli

  • 分子量

    70kDa (Reducing)

  • 纯度

    ＞95% by SDS-PAGE and HPLC

  • 活性

    5 U/ul

  • 标记

    Unconjugated

  • 标签

    His Tag

  • 性状

    Liquid

  • 缓冲体系

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

  • 溶解方法

    /

  • 储存条件

    Store at -25 ~ -15℃ for 2 years

  • 文献引用

    [1] Zhao, Guojie , et al. "Realizing directional cloning using sticky ends produced by 3′-5′ exonuclease of Klenow fragment." Journal of Biosciences 38.5(2013):857-866.
    [2] Olsen, Tivoli J. , et al. "Electronic Measurements of Single-Molecule Processing by DNA Polymerase I (Klenow Fragment)." Journal of the American Chemical Society 135.21(2013):7855-7860.

背景介绍

  • The Klenow Fragment, is a large fragment of E.coli. DNA polymerase I. It retains the 3'→5' exonuclease activity of DNA polymerase I, but lacks the 5'→3' exonuclease activity of the intact DNA polymerase I. The 3'→5' exonuclease activity of Klenow Fragment ensures accurate proofreading when synthesizing DNA. It is used to fill in the 5'overhang ends of double-stranded DNA; and double-stranded DNA 3'overhang flattening (also called trimming). It can also be used for the synthesis of the second strand of cDNA or the synthesis of the second strand of site-specific mutation reaction.

产品组分

  • Storage Solution: 5 U/ul Klenow Fragment、25 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.4 @ 25°C 10*Reaction Buffer: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT (pH 7.9 @ 25°C)

操作步骤

  • 1. DNA should be dissolved in 1*Reaction Buffer or T4 DNA Ligase Reaction buffer and supplemented with 33 μM each dNTP.

    2. Add 1 unit of DNA Polymerase I Large (Klenow) Fragment per microgram DNA.

    3. Incubate for 15 minutes at 25°C.

    4. Stop reaction by adding EDTA to a final concentration of 10 mM and heating for 20 minutes at 75°C.

注意事项

  • 1. Due to the 3´→5´ exonuclease activity of the enzyme, increasing the reaction temperature, adding too much enzyme, not adding dNTP or too long reaction time will lead to the formation of the dented end.

    2. Please avoid repeated freeze-thaw cycles

酶活定义
  • One unit is defined as the amount of enzyme required to incorporate 10 nmol of dNTP into acid insoluble substances at 37 ° C for 30 minutes.

  • 生物活性

    • The experiment was designed to synthesize two complementary DNA Oligos. Small DNA fragments with 5 'sticky ends were synthesized by annealing, and the sticky ends were completed by Klenow fragment to form flat ends. 20% Native PAGE glue (which can distinguish the size of 0.1 ~ 100bp) was used for electrophoresis, and the enzyme activity was verified. As can be seen from the result, the dsDNA fragment at the sticky end is mended by Klenow fragment, which has a larger molecular weight, and the electrophoretic band position is above the DNA fragment with the sticky end. The results show that UA070056-Klenow fragment has the same effect as a competing product N.
      Lane 1 Negative Control
      Lane 2 competing product N 0.44μg
      Lane 3 competing product N 0.22μg
      Lane 4 UA070056-Klenow fragment 0.44μg
      Lane 5 UA070056-Klenow fragment 0.22μg
      Lane 6 UA070056-Klenow fragment 0.42μg
      Lane 7 UA070056-Klenow fragment 0.21μg

  • 电泳

    • 1μg (R: reducing condition, N: non-reducing condition).

  • 反相高效液相色谱(RP-HPLC)

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