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Ammonium Acetate (5 M), RNase-free, 500 mL - FAQs

查看更多产品信息 Ammonium Acetate (5 M), RNase-free - FAQs (AM9070G, AM9071)

3 个常见问题解答

使用哪种盐可以沉淀总RNA？每种盐的优势是什么？这些盐是否需要使用乙醇？

可使用以下3种盐：

•硫氰酸胍，需要乙醇。硫氰酸胍是分离RNA的常用试剂，我们特别推荐将其用于核糖核酸酶活性较高的组织，如胰腺或脾脏。
•醋酸铵，需要乙醇。醋酸铵可用于减少不必要的dNTPs和寡糖共沉淀。但是，纯化的核酸如果后续要使用T4多核苷酸激酶处理的话，不能使用醋酸铵，因为铵离子会抑制T4多核苷酸激酶。
•氯化锂，不需要乙醇。LiCl能有效沉淀RNA而不能有效沉淀蛋白质或DNA，而且也不会沉淀未掺入的游离核苷酸。

What salts can be used to precipitate total RNA? What are the advantages of each? Do they require ethanol?

The three salts that can be used are:

- Guanidine thiocyanate, which requires ethanol. Guanidine thiocyanate is a common agent used for isolating RNA and we recommend it especially for tissues high in ribonuclease activity, such as the pancreas or spleen.
- Ammonium acetate, which requires ethanol. Ammonium acetate is useful when reducing coprecipitation of unwanted dNTPs and oligosaccharides. However, it should not be used when the nucleic acid will be phosphorylated using T4 polynucleotide kinase, since this enzyme is inhibited by ammonium ions.
- Lithium chloride, which does not require ethanol. LiCl is very effective in precipitating RNA but does not efficiently precipitate protein or DNA. It also does not precipitate unincorporated nucleotides.

How can I concentrate DNA solution using ethanol precipitation?

Ethanol precipitation is frequently used for concentration of DNA solutions and for removal of protein, salt, and unincorporated nucleotides. The two most common protocols use either 0.3 M sodium acetate (0.1 volume of 3 M) or 2.5 M ammonium acetate (0.5 volume of 7.5 M), along with 2 to 2.5 volumes of ethanol. Studies at Thermo Fisher Scientific (1, 2) have shown these two salts to be equally effective for recovery of small amounts of DNA from small volumes and for removal of unincorporated nucleotides from labeling reactions.

DNA was found to precipitate readily with room temperature ethanol and room temperature centrifugation. For DNA concentrations >0.1 µg/mL, no incubation period is required. For improved recovery of DNA from dilute solutions (10 ng/mL), overnight incubation in the ethanol and extended (30 min) centrifugation is recommended. Addition of ammonium acetate to 2.5 M (without ethanol) has also been shown to be effective in precipitating proteins while leaving the DNA in solution (2).

1. Zeugin, J.A. and Hartley, J.L. (1985) FOCUS 7:4, 1.

2. Crouse, J. and Amorese, D. (1987) FOCUS 9:2, 3.