How Ethanol precipitation works

Ethanol precipitation is a common method used to purify and/or concentrate RNA, DNA and polysaccharides from aqueous solutions by adding ethanol. This forces the nucleic acid to precipitate out of solution.

DNA precipitation happen with the correct concentration of positive ions is present in solution. Too much salt will result in a lot of salt co-precipitating with DNA, too little will result in incomplete DNA recovery.

DNA is polar due to its highly negative charged phosphate (PO3-). This polarity, based on the principle of "like dissolves like", makes it soluble in water, which is also highly polar. Salt (mostly sodium acetate Na+ [CH3COO]-.) is added in protocol to neutralize the charges on sugar phosphate backbone. As the mechanism suggests, the solution has to contain positive ions for precipitation to occur; usually Na+, NH4+ or Li+ plays this role. 


Salt breaks down into positively charged sodium ions neutralizing the negative charge on the PO3- groups on Nucleic acids, creating less hydrophilic molecules, and therefore much less soluble in water. Ethanol is much less polar than water, with a dielectric constant of 24.3 at 25 °C. Adding ethanol to solution disrupts the screening of charges by water (Coulomb's Law applies here).

If enough ethanol is added , the electrical attraction between (PO3-) groups and positive Na+ ions present in solution becomes strong enough to form stable ionic bonds. This make the nucleic acid less hydrophilic, causing it to drop out of solution and DNA precipitate . 

Optimal incubation time depends on the length and concentration of DNA. Smaller fragments and lower concentrations will require longer times to achieve the same recovery. During incubation DNA, some salts will precipitate from solution, which is collected by centrifugation After centrifugation the supernatant solution is removed, leaving a pellet of crude DNA. Whether the pellet is visible depends on the amount of DNA and on its purity (dirtier pellets are easier to see) or the use of co-precipitants. 

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