Ethanol Precipitation Protocol
Reagents
- 3M Sodium Acetate, pH 5.2 (CAS 127-09-3)
- Ethanol, 200 Proof (CAS 64-17-5)
- Ethanol, 70%
- 10 mM Tris-HCl Buffer Solution or TLE Buffer Solution
Procedure
- Add 1/10 volume of 3 M sodium acetate relative to sample volume
- Add 3X volume of ethanol relative to sample volume including the added sodium acetate
- Incubate at room temperature for at least 15 minutes
- Centrifuge at 16,100 rcf for 30 minutes
- Carefully decant the supernatant, making sure the DNA pellet (which may not be visible) is not discarded
- Wash with a volume of 70% ethanol equivalent to or greater than the final volume achieved after step 2
- Centrifuge at 16,100 rcf for 15 minutes
- Decant supernatant
- Briefly spin tube to collect residual ethanol
- Pipette and discard remaining ethanol being sure to not disturb the DNA pellet
- Dissolve pellet in TLE buffer solution or in 10 mM Tris-HCl if downstream applications are affected by the presence of EDTA.
Tips
- If working with small quantities of DNA where there may not be a visible pellet, orient the microcentrifuge tubes so the the hinge faces outward. The pellet should form on the hinge side of the tube and can thus be avoided when pipetting.
- DNA yields can be most effectively increased by increasing the initial incubation time in step 3. Lowering the incubation temperature and temperature of solutions can increase yields, especially for smaller fragments, but may also increase the coprecipitation of salts. Longer centrifugation times can also help but if heat builds up in the centrifuge this may be problematic. A refrigerated centrifuge run at 4°C can help reduce the temperature and increase yields.