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.
- Sodium Acetate
- Ammonium Acetate
- Lithium Chloride
- Sodium Chloride
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.
- Yeast tRNA – 10–20 μg⁄mL
- Salmon Sperm – 10–20 μg⁄mL
- Glycogen – 50–150 μg⁄mL
- Linear Polyacrilamide – 10–20 μg⁄mL
Ethanol Precipitation – P. ZUMBO, DEPARTMENT OF PHYSIOLOGY & BIOPHYSICS
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