DNA 聚合酶 I 大 (Klenow) 片段
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DNA 聚合酶 I 大 (Klenow) 片段

DNA 聚合酶 I 大 (Klenow) 片段是一种 DNA 聚合酶，缺乏完整 DNA 聚合酶了解更多信息
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货号数量
18012021100 单位
18012039500 单位
18012096
又称 18012-096
2 x 500 单位
货号 18012021
价格（CNY)
3,438.00
100 units
添加至购物车
数量:
100 单位
价格（CNY)
3,438.00
100 units
添加至购物车
DNA 聚合酶 I 大 (Klenow) 片段是一种 DNA 聚合酶，缺乏完整 DNA 聚合酶 I 的 5' 至 3' 核酸外切酶活性，但具有 5' 至 3' DNA 聚合酶和 3' 至 5' 核酸外切酶活性。

应用：
填充 5´ 突出端 (1)。通过随机引物标记方法合成探针 (2)。对单链和双链 DNA 进行测序 (3)。定点诱变。

来源：
从在质粒上表达 Klenow 片段的大肠杆菌中纯化获得。

性能和质量检测：
SDS-PAGE 纯度；单链和双链内切脱氧核糖核酸酶和自引发测定；在填充反应中评估性能。

单位定义：
1个单位的酶量可以在 37°C 下使用模板•引物在 30 min 内向酸沉淀材料掺入 10 nMol 总脱氧核糖核酸。
仅供科研使用。不可用于诊断程序。
规格
描述DNA 聚合酶
外切核酸酶活性3' 至 5'
热启动否
聚合酶DNA 聚合酶 I
数量100 单位
运输条件经批准可置于湿冰或干冰上运输
Unit Size100 units
内容与储存
DNA 聚合酶 I 大 (Klenow) 片段配有1小瓶 10X REact™ 2 缓冲液 [500 mM Tris-HCl (pH 8.0)、100 mM
MgCl2、500 mM NaCl] 和1小瓶稀释缓冲液。储存在 -20°C 下。

常见问题解答 (FAQ)

我想将一个含5’或3’突出端的分子转变成平末端分子，应使用什么酶？这些酶有何区别？

T4 DNA聚合酶和大肠杆菌DNA聚合酶的Klenow片段，均可将突出端分子转变成平末端分子。但T4 DNA聚合酶的3’—5’核酸外切酶活性比Klenow更强。

What enzymes can I use to convert a molecule with a 5' or 3' overhang to a blunt molecule? What is the main difference between them?

T4 DNA polymerase and Klenow fragment of E.coli DNA polymerase can both convert overhangs to blunt molecules. The 3' - 5' exonuclease activity of the T4 DNA polymerase is much more efficient than Klenow.

What conditions are optimal for fill-in reactions using Klenow (Large fragment of DNA Polymerase)?

Klenow can be used to "fill in" 5' overhangs of double-stranded DNA fragments using common restriction endonuclease buffers. We recommend REact 2 buffer (1X concentration is 50 mM Tris-HCl pH 8, 50 mM NaCl, 10 mM MgCl2) , but other buffers will also work.

Fill-in Reaction Conditions:

1. Dilute Large Fragment of DNA Polymerase I to 0.5 U/µL with Klenow Dilution Buffer.
2. To a 1.5-mL microcentrifuge tube on ice, add: 10X REact 2 Buffer - 3 µL, 0.5 mM dATP - 1 µL, 0.5 mM dCTP - 1 µL, 0.5 mM dGTP - 1 µL, 0.5 mM dTTP - 1 µL, DNA 0.5-1 µg, Large fragment of DNA Polymerase I - 1 µL, Autoclaved distilled water to 30 µL.
3. Mix gently and centrifuge briefly to bring the contents to the bottom of the tube.
4. Incubate at room temperature for 10-15 minutes or 20 minutes on ice.
5. Terminate fill-in reaction by phenol extraction.

To label the DNA fragment, use 1-2 µL of [alpha-32P]dNTP (400 Ci/mmol, 10 mCi/mL) (24-48 pmoles) instead of the corresponding cold dNTP.

Note: Thermo Fisher Scientific also offers an Exo minus Klenow, which is provided with its own buffers.

Is there anything to prevent AmpliTaq Gold DNA polymerase from extending from the 3’ end of a TaqMan probe in a 5’ nuclease assay?

Yes. There is a phosphate group on the 3' end of all TaqMan probes that prevents such extension.

How does AmpliTaq Gold DNA Polymerase differ from AmpliTaq DNA Polymerase?

AmpliTaq Gold DNA Polymerase is a modified form of AmpliTaq DNA Polymerase that contains a proprietary chemical (or so-called hot start molecule) bound to the enzyme's active site. In order to activate the AmpliTaq Gold DNA Polymerase fully, we recommend an initial activation step of 95 degrees C for 10 min when using GeneAmp 10X PCR Buffer I and/or GeneAmp 10X PCR Buffer II and Mg in one of our thermal cyclers. When using GeneAmp 10X PCR Gold Buffer, activation time can be reduced to 5 minutes. Once activation is complete, you can proceed with your standard PCR cycling program (denaturing, annealing, extension, etc).

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