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Ethanol Precipitation of DNA and RNA: How it works

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Ethanol precipitation is a commonly used technique for concentrating and de-salting nucleic acids (DNA or RNA) preparations in aqueous solution.

The basic procedure is that salt and ethanol are added to the aqueous solution, which forces the precipitation of nucleic acids out of solution. After precipitation the nucleic acids can then be separated from the rest of the solution by centrifugation. The pellet is washed in cold 70% ethanol then after a further centrifugation step the ethanol is removed, and the nucleic acid pellet is allowed to dry before being resuspended in clean aqueous buffer. So how does this work?

ethanol precipitation is all about solubility

Ethanol precipitation of nucleic acids is all about solubility…

First we need to know why nucleic acids are soluble in water. Water is a polar molecule – it has a partial negative charge near the oxygen atom due the unshared pairs of electrons, and partial positive charges near the hydrogen atoms (see the diagram on the right).

Because of these charges, polar molecules, like DNA or RNA, can interact electrostatically with the water molecules, allowing them to easily dissolve in water. Polar molecules can therefore be described as hydrophilic and non-polar molecules, which can’t easily interact with water molecules, are hydrophobic. Nucleic acids are hydrophilic due to the negatively charged phosphate (PO3-) groups along the sugar phosphate backbone.

The role of the salt in ethanol precipitation…

Ok, so back to the protocol. The role of the salt in the protocol is to neutralize the charges on the sugar phosphate backbone. A commonly used salt is sodium acetate. In solution, sodium acetate breaks up into Na+ and [CH3COO]-. The positively charged sodium ions neutralize the negative charge on the PO3- groups on the nucleic acids, making the molecule far less hydrophilic, and therefore much less soluble in water.

The role of the ethanol…

The electrostatic attraction between the Na+ ions in solution and the PO3- ions are dictated by Coulomb’s Law, which is affected by the dielectric constant of the solution. Water has a high dielectric constant, which makes it fairly difficult for the Na+ and PO3- to come together. Ethanol on the other hand has a much lower dielectric constant, making it much easier for Na+ to interact with the PO3-, shield it’s charge and make the nucleic acid less hydrophilic, causing it to drop out of solution.

The role of temperature in ethanol precipitation…

Incubation of the nucleic acid/salt/ethanol mixture at low temperatures (e.g. -20 or -80C) is commonly cited in protocols as necessary in protocols. However, according to Maniatis et al (Molecular Cloning, A Laboratory Manual 2nd Edition… 2nd edition?? – I need to get a newer version!), this is not required, as nucleic acids at concentrations as low as 20ng/mL will precipitate at 0-4C so incubation for 15-30 minutes on ice is sufficient.

The wash step with 70% ethanol…

This step is to wash any residual salt away from the pelleted DNA.

A few tips on ethanol precipitation…

  • Choice of salt
    • Use Sodium acetate (0.3M final conc, pH 5.2) for routine DNA precipitations
    • Use Sodium chloride (0,2M final conc) for DNA samples containing SDS since NaCl keeps SDS soluble in 70% ethanol so it won’t precipitate with the DNA.
    • Use Lithium Chloride (0.8M final conc) for RNA. This is because 2.5-3 volumes of ethanol should be used for RNA precipitation and LiCl is more soluble in ethanol than NaAc so will not precipitate, but beware – chloride ions will inhibit protein synthesis and DNA polymerase so LiCl is no good for RNA preps for in vitro translation or reverse transcription. In these cases, use NaAc.
    • Use Ammonium acetate (2M final conc) for the removal of dNTPs, but do not use for preparation of DNA for T4 polynucleotide kinase reactions as ammonium ions inhibit the enzyme.
  • To increase the yield in precipitations of low concentration or small nucleic acid pieces (less than 100 nucleotides)
    • Add MgCl2 to a final concentration of 0.01M
    • Increase the time of incubation ice before centrifugation to 1 hour.

This explanation should bring  you up to speed on how the ethanol precipitation works. If you want to learn more about the ins and outs of ethanol precipitation and other DNA cleanup approaches, you might want to check out these…

More articles on Ethanol precipitation

150 Comments

  1. Poulami on March 30, 2017 at 1:56 pm

    I had mistakenly added sodium acetate at pH 7.0. How can I troubleshoot?

  2. Nia on February 22, 2017 at 5:20 am

    Thank you so much!!

  3. Hans on October 21, 2016 at 1:24 pm

    Hi,
    I want to get rid of TE buffer in a soil DNA extract and thought of precipitating DNA with ethanol. Does anyone know how much of the original DNA can be recovered/will be lost?

    Thanks!

  4. Monika on February 7, 2016 at 12:21 pm

    How can we remove the ethanol from the eluted DNA in case if it still remains in it ?

    • Habeeb on February 18, 2016 at 5:55 am

      Hello,
      I think leaving the eluted DNA in RT for 15-20 minutes will remove the remaining EtOH.

      • Kyle on February 22, 2017 at 2:51 am

        If you want to be absolutely certain you can even speedvac it dry (or just leave it on a heat block, depending on your volume). You’ll be left with a pellet of DNA and the dried salts from the buffer to be resuspended in water.

  5. Volker Baumann on January 31, 2015 at 3:47 pm

    Hello, I just have a question to the part: “To increase the yield in precipitations of low concentration or small nucleic acid pieces (less than 100 nucleotides)

    Add MgCl2 to a final concentration of 0.01M”

    Is there somewhere a reference why 0.01M MgCl2 is supporting RNA <100bp for precipitation?

  6. yoobios on November 4, 2013 at 4:46 am

    Thanks to all authors, I’ve got a useful information about EtOH precipitation.
    But I still have one question about “Choice of salt”.
    1.How can each type of salt differ the yield?
    2.What is the different between NaOAc and ammonium acetate’s action on DNA?
    3.How can ammonium acetate remove dNTPs ? Can’t NaOAc remove them? and why?

  7. user-68239 on June 7, 2013 at 4:06 am

    HiI am a new member. thanks for ur great site

  8. vision_leaks on June 6, 2013 at 9:35 am

    Very nice article!
    For the salt solutions there are different pH recomendations.
    Could you explain which pH is ideal for RNA and which for DNA and why that is?

    Cheers Anja

  9. JFB on November 27, 2012 at 7:36 pm

    Nice article and also helpful comments. I’m still a bit confused about one point: if salt is so important for neutralizing the negative charge on the RNA/DNA so that a pellet forms during the first centrifugation, then why does a pellet still form during the 2nd spin with 70% EtOH after the salt has been washed away?

    • carson clark on November 9, 2017 at 4:55 am

      exactly my question. I guess the ethanol is still nonpolar enough to precipitate remaining DNA? I don’t know why the salt is necessary then…

    • Jeni on April 9, 2019 at 5:30 am

      the pellet is already formed and remains intact during the ethanol wash.

      • vaisak on August 13, 2019 at 1:40 pm

        Not necessarily. Even if you redissolve and then centrifuge, you will get the pellet back from EtOH only solution.

      • GOPINATH S on October 25, 2019 at 6:18 am

        I mistakenly added ethanol first and sodium acetate next… Does this affects the preparation of DNA.

  10. uday_pratap on June 19, 2012 at 8:23 am

    Hey Nick..Hats off for u man….
    This is quite basic things…..i dont know.from where do u get these knowledge bro..?
    very thanks….

    • csequeira on November 12, 2012 at 10:17 pm

      I have a question maybe you can answer. Why is isopropanol more effective at precipitating nucleic acids than ethanol?

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