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Thursday, February 13, 2014

Ethanol Precipitation of DNA - Principle and Methods


Ethanol Precipitation is a method for purifying or concentrating DNA/RNA from aqueous solutions using Ethanol as anti-solvent.The precipitation is accompanied by the addition of salt and ethanol to a solution containing DNA or RNA.In presence of monovalent cations (eg: Na+) ethanol efficiently precipitates nucleic acids. The precipitate can be collected by centrifugation.






Ethanol Precipitation of DNA - Principle


DNA surrounded by water molecules
 (Hydration Shell) in solution
DNA has net negative charge because of its phosphate backbone. sodium acetate (CH3COONa) which is most widely used in the ethanol precipitation is a carboxilate salt, carboxilate salts contain a carboxilate anion and a positively charged metal ion. when salt is added to a solution containing nucleic acids, the salt dissociates to CH3COO- and Na+.

When the cations and negatively charged nucleic acid backbone interact, nucleic acids are neutralized, therefore no longer dissolve in water and precipitate out of solution.
Ethanol which has lower dielectric constant compared to water increases the interaction of the salt and the Coulomb force of attraction between the cations and the negatively charged nucleic acid backbone (that is, the resistance from the solvent’s electric field sufficiently diminishes to permit efficient interaction; the solvation shells surrounding the solute’s charges depletes).

Ethanol also induces conformational changes to the nucleic acid structure; the repulsive forces between the negatively charged phosphates with a nucleic acid polymer are so diminished that inter-helical interactions are possible, allowing the nucleic acids to aggregate.

Materials
  • 3 M sodium acetate pH 5.2 or 5 M ammonium acetate 
  • DNA 
  • 100% ethanol 
Procedure 
  • Measure the volume of the DNA sample. 
  • Adjust the salt concentration by adding 1/10 volume of sodium acetate, pH 5.2 (final concentration of 0.3 M) or an equal volume of 5 M ammonium acetate (final concentration of 2.0 to 2.5 M). These amounts assume that the DNA is in TE only. If DNA is in a solution containing salt, adjust salt accordingly to achieve the correct final concentration. Mix well. 
  • Add 2 to 2.5 volumes of cold 100% ethanol (calculated after salt addition). Mix well. 
  • Spin a maximum speed in a microfuge for 10–15 min. 
  • Carefully decant supernatant. 
  • Add 1 mL 70% ethanol and mix spin briefly. 
  • Carefully decant supernatant. 
  • Air dry or briefly vacuum dry pellet. 
  • Resuspend pellet in the appropriate volume of TE or water.
Types of Salts used for Ethanol Precipitation of DNA
  • Sodium Acetate
  • Ammonium Acetate
  • Lithium Chloride
  • Sodium Chloride
Carriers or Co-Precipitants

Carriers or Co-precipitants are substances which improves the nucleic acid target recovery.They are insoluble in ethanol or isopropanol solutions, and they form a precipitate that helps to trap nucleic acids by bulk. During centrifugation, carriers form a visible pellet, which aids in removing the supernatant without
perturbing the nucleic acid pellet. Precipitations with carriers from diluted solutions of nucleic acids are also quantitatively more efficient than precipitations without carriers.

List of carriers and their final concentration used in Ethanol Precipitation of DNA
  • Yeast tRNA - 10–20 μg⁄mL
  • Salmon Sperm - 10–20 μg⁄mL
  • Glycogen - 50–150 μg⁄mL
  • Linear Polyacrilamide - 10–20 μg⁄mL
Role of Temperature on DNA Precipitation

As the temperature of an alcohol-aqueous solution decreases, its dielectric constant increases. Therefore, in principle, at increasingly chilled temperatures, precipitation efficiency should diminish. Moreover, as the temperature decreases, the viscosity of the solution increases, which retards the movement of the nucleic acid aggregate, especially if the aggregate is small. In addition, since solubility decreases at lower temperatures, more salts will begin to co-precipitate with the nucleic acids at lower temperatures. Therefore, incubation at temperatures below 0°C is counter-productive and not recommended.

Short Protocols in Molecular Biology, 5th Edition

References

Ethanol Precipitation - P. ZUMBO, DEPARTMENT OF PHYSIOLOGY & BIOPHYSICS
Internet Sources