Published on February 18, 2014
Methods & Principle Ethanol Precipitation of DNA www.technologyinscience.blogspot.com
• Ethanol Precipitation is a method for purifying or concentrating DNA/RNA from aqueous solutions using Ethanol as anti-solvent. • In presence of monovalent cations (eg: Na+) ethanol efficiently precipitates nucleic acids. The precipitate can be collected by centrifugation.
Ethanol Precipitation of DNA Principle • When the cations from salt and negatively charged nucleic acid backbone interact, nucleic acids are neutralized, therefore no longer dissolve in water and precipitate out of solution. • Ethanol Improves the interaction of salt with the DNA.
Ethanol Precipitation of DNA Principle
Types of Salts used for Ethanol Precipitation of DNA • • • • Sodium Acetate Ammonium Acetate Lithium Chloride Sodium Chloride
Carriers or Co-Precipitants • • • • Yeast tRNA - 10–20 μg⁄mL Salmon Sperm - 10–20 μg⁄mL Glycogen - 50–150 μg⁄mL Linear Polyacrilamide - 10–20 μg⁄mL
Materials Required for Precipitation • 3 M sodium acetate pH 5.2 or 5 M ammonium acetate • DNA • 100% ethanol
Procedure for DNA Precipitation • • • • • • • • • 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.
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