Ammonium Acetate (5 M), RNase-free

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.

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.