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ethanol-precipitation.md

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Title: Ethanol precipitation Authors: Brant C. Faircloth
Address1: Department of Ecology and Evolutionary Biology, University of California, Los Angeles, CA 90095, USA
Contact: Brant C. Faircloth
ContactEmail: brant.faircloth at gmail dot com
Copyright: 2010 Brant Faircloth
This work is licensed under a Creative Commons Attribution License.
http://creativecommons.org/licenses/by/3.0/us/
Keywords: DNA extraction, precipitation, ethanol precipitation

Summary

This protocol provides a method for precipitating DNA from a solution using ethanol and a binding salt (typically sodium acetate, but sometimes ammonium acetate). EtOH precipitation is used for a number of reasons: to concentrate DNA in the absence of a speedvac, to "clean" DNA following PCR procedures (amplification, sequencing), and as a part of many DNA extraction procedures.

The mechanics of ethanol precipitation derive from the fact that strands of DNA in solution (water) are kept from binding to each other due to the solvent properties of water. When ethanol is added to the water, it basically reduces the ability of water to keep the charged phosphate backbones of DNA strands from binding to one another - thus, in the presence of ethanol, DNA precipitates from solution. We add a salt to the solution as a source of ions facilitating the precipitation of DNA.

You may wish to use alternative salt solutions (ammonium acetate, sodium chloride) in certain circumstances (see Sambrook and Russell 2001). For instance, ammonium acetate is often used to reduce the precipitation of contaminants that "travel along" with the precipitated DNA - e.g. plant DNA extraction using CTAB.

The articles by both Zeugin and Hartley (1985) and Crouse and Amorese (1987) generally indicate that incubations at 0 C are preferable to those at -70; increased incubation times (at 0 C) have negligible effect on the amount of DNA precipitated; and centrifugation longer than 15-20 minutes has a moderate effect on amount of DNA recovered, assuming that the DNA concentration is high to begin with (in other words, more dilute samples might benefit from longer spins).

Goal

To concentrate/clean DNA in solution

Main references

  • Sambrook J, Russell DW (2001). Molecular cloning: A laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, NY, USA.
  • Zeugin JA, Hartley JL (1985). Ethanol Precipitation of DNA. Focus 7:1-2.
  • Crouse J, Amorese D (1987). Ethanol Precipitation: Ammonium Acetate as an Alternative to Sodium Acetate. Focus 9:3-5.

Inputs

  • DNA in solution (typically H2O or TE/TLE)

Outputs

  • Clean, concentrated DNA

Materials needed

  1. 3 M sodium acetate
  2. 95% Ethanol
  3. 70% Ethanol
  4. [optional] 10 M ammonium acetate
  5. [optional] 5 M sodium chloride

Detailed steps

  1. Add 1/10th volume of 3 M sodium acetate to tube containing DNA in solution. [optional - you can use 1/5th volume of ammonium acetate or 1/20th volume NaCl - when DNA solution contains SDS]
  2. Add 2 volumes of cold 95-100% ethanol to tube containing DNA in solution. [optional - you can use one volume of isopropanol in place of ethanol, but ethanol is preferred]
  3. Incubate 10 m on ice (or in -20)
  4. Place carefully in centrifuge with hinges facing "out". You do not need a refrigerated centrifuge
  5. Centrifuge 5 m, full speed (~14,000 RPM)
  6. Discard the ethanol (dump into hazmat container)
  7. Add 750 uL 70% ethanol
  8. Centrifuge 1 m, full speed (~14,000 RPM)
  9. Discard the ethanol and allow the tubes to dry (invert on kimwipe)
  10. Rehydrate DNA pellet in solution (TLE) and volume of choice