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Molecular Pathology

DNA Purification

Ethanol Precipitation / Salting-Out


Author: Rodney E. Shackelford, D.O., Ph.D. (see Reviewers page)
Revised: 14 April 2012, last major update April 2012
Copyright: (c) 2008-2012, PathologyOutlines.com, Inc.

Definition
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● Uses high aqueous salt concentrations to separate proteins and other cellular contaminants from nucleic acids
● Alcohol is added to aqueous solution and nucleic acids are removed by centrifugation

Theory
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● DNA is polyanionic due to large number of negatively charged phosphate groups in its phosphodiester-linked backbone; dissolves well in highly polar/high dielectric constant solvents like water
● DNA typically does not form ionic bonds with dissolved cations in aqueous solutions, as water molecules form a hydration shell around most ions preventing ionic bond formation and DNA precipitation; an exception is calcium cations, which rapidly bind and precipitate phosphate groups out of aqueous solutions, forming a precipitate that is difficult to resolubilize
● Common cations used in DNA precipitation include Na+, NH4+, Li+
● Ethanol and isopropyl alcohols are less polar than water, having a dielectric constant of 24.3 (ethanol) vs. 80.1 for water
● Adding ethanol to an aqueous DNA solution to a concentration of 64% or greater allows the phosphodiester DNA backbone to form ionic bonds with cations, resulting in DNA precipitation

Technique
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● For optimal DNA precipitation, the concentration of cations must be sufficient to form ionic binds with the DNA, but not have so much salt that it precipitates out of solution by itself; conversely, too few cations results in poor DNA precipitation and a low DNA recovery
● Optimal DNA precipitation is achieved at room temperature; storage of precipitation mix at low temperatures lowers precipitation efficiency
● Low precipitation temperatures are often used because they lower nucleic acid degradation from trace nucleases in solution
● In general, shorter DNA fragments require longer periods to precipitate
● DNA recovery is facilitated by higher molecular weight DNA and high DNA concentrations
● DNA precipitation is facilitated by centrifugation, usually at 12,000gs and higher, at 0-4 C
● The resulting DNA pellet is washed, often with 70% ethanol or isopropyl alcohol to remove remaining salts
● Often alcohol precipitation purification is followed by other purification techniques, such as organic extraction or CsCl centrifugation to achieve greater nucleic acid purity

End of Molecular Pathology > DNA purification > Ethanol Precipitation / Salting-Out


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