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- 详细信息
- 文献和实验
- 技术资料
- 保存条件:
低温
- 英文名:
Uracil DNA Glycosilase
- 规格:
200U×5
| Synonyms | Uracil DNA Glycosilase, Uracil DNA Glycosylase, UNG. |
| Description | E.Coli Uracil DNA Glycosilase (UNG) catalyses the release of free Uracil from Uracil-containing DNA. UNG efficiently hydrolyzes uracil from signle-stranded or double-stranded DNA, but not from oligomers (6 fewer bases). |
| Source | Escherichia Coli strain that carries the UNG gene from E.coli. |
| Physical Appearance | Sterile Filtered colorless solution. |
| Formulation | 10mM Tris-Hcl (pH-7.4 at 25°C), 50mM KCl, 1mM DTT, 0.1mM EDTA, 0.1 mg/ml BSA and 50% glycerol. |
| Stability | Uracil DNA Glycosilase although stable at 15°C for 1 week, should be stored desiccated below -18°C. Please prevent freeze-thaw cycles. |
| Applications | Glycosilase mediated single nucleotide polymorphism detection (GMPD). Site-directed mutagenesis. As a probe for protein-DNA interaction sudies. Rapid and efficient cloning of PCR products. Elimination carry-over contamination in PCR. |
| Specific Activity | The Specific Activity was found to be 5U/?l. |
| Unit Definition | 1 Unit is defined as the amount of UDG enzyme that catalyzes release of 60pmol of Uracil per minute from double-stranded, uracil-containing DNA in a total reaction volume of 50µl in 30 minutes at 37°C in 1X Uracil DNA Glycosilase Reaction Buffer with 1 Unit of Uracil DNA Glycosylase and 0.2µg [3H]-Uracil DNA (104- 105 cpm/µg). |
| 10X UNG Reaction Buffer | 200mM Tris-HCl (pH 8.0 at 25°C), 10mM DTT and 10mM EDTA. |
| Reaction Conditions | 1X UNG Reaction Buffer, incubate at 37°C. UNG is active over a broad pH rabge with an optimum at pH-8.0, doesn't require divalent cation, and is inhibited by high ionic strength (>200mM). The abasic sites formed in DNA by UNG may be cleaved by heat, alkali-treatment or endonucleases that cleave specifically at abasic sites. |
| Usage | Prospec's products are furnished for LABORATORY RESEARCH USE ONLY. They may not be used as drugs, agricultural or pesticidal products, food additives or household chemicals. |
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文献和实验」和「扩增区」分割开 PCR 体系配置和 PCR 扩增实验。如果不具备这样的条件,首先要避免荧光定量 PCR 实验结束之后开盖,以防扩增产物形成气溶胶对后续实验造成污染。少量意外开盖不能避免时,可以使用含有 UNG 酶(uracil-N-glycoslyase, 尿嘧啶 DNA 糖基化酶)的扩增预混液。如果在 PCR 反应中以 dUTP 代替 dTTP 参入到 PCR 产物中,形成了含有 dUTP 的扩增产物,UNG 酶能选择性断裂单链和双链 DNA 中 U 碱基的糖苷键, 降解 PCR 扩增
特异性反应。应用荧光素标记抗体使之与预先已固定在DEAE膜上的样品DNA特异性结合,对DEAE膜上的荧光素进行扫描得到5mC的水平,其荧光素强度与5mC水平成正比。Oakeley等1997年[23]报道了这种方法。这种方法需要精密的仪器。 2.1.4 氯乙醛法 Oakeley等1999年[24]首先描述了这种使用氯乙醛和荧光标记的方法。首先,将DNA经重亚硫酸盐处理使未甲基化的胞嘧啶全部转变为尿嘧啶,而甲基化的胞嘧啶保持不变(Frommer等1992年)[25],然后经过银或色谱柱去除DNA链
和耐药性相关突变。 3.4 DNA 甲基化常用检测方法 近年来涌现出很多 DNA 甲基化的检测技术,目前依据对目标 DNA 的预处理手段可将甲基化检测技术分为 3 类: 3.4.1 基于限制性酶切预处理的甲基化检测技术 这种方法是利用甲基化敏感性限制性内切酶对甲基化区域不切割的特性,将 DNA 消化为不同大小的片段后再进行分析。常用的经典酶对是 Hpa II - Msp I(识别序列 CCGG )。随后进行 Southern 或 PCR 扩增分离产物,以明确目标片段的甲基
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