The Basics: How Ethanol Precipitation of DNA and RNA Works
Ethanol precipitation is a commonly used technique for concentrating and de-salting nucleic acid (DNA or RNA) preparations in aqueous solution. The basic procedure is that salt and ethanol are added to the aqueous solution, which forces the nucleic acid to precipitate out of solution. The precipitated nucleic acid 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?
A bit 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…
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…
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 nucleic acid 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.
If you have anything to add, please feel free to leave a comment!
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Nick Oswald
Nick is a molecular biologist-turned-publisher. After a PhD in Developmental Biology and an eclectic seven years in biotech he is now Editorial Manager of Neuroendocrinology and the founder and Editor-In-Chief of Bitesize Bio. . You are welcome to connect with Nick on LinkedIn
Phosphate is PO4 3-
Should it not be PO4 3- everywhere you have written PO3-?
Thanks
Ok, Just a basic question…at school in my lab we were required to keep the ethanol at a low temperature before using it and my SI said it was due to evaporation although she seemed like she was just taking a crack at it rather than actually giving me a legit answer.
So,
1. Why is the ethanol kept at a low temperature rather than at room temperature?
2. Would freezing the sample (a calf thymus, not a plant)affect the ability to extract the DNA from the cells?
Jason,
The ethanol is kept at a low temperature to reduce the solubility of the DNA so that it precipitates out more effectively. Freezing the thymus sample might actually make it easier to remove the DNA by breaking up the tissue a bit so that homogenization is easier.
Hope that answers your question.
Nick
Thanks, this info is awesome.
Hi Nick
I have a question. I am trying to precipitate DNA using the commonly used ETOH method. Initially I added 0.1 vol of NaOAc (3M pH 5.2)and 2.5 volume EtOH (100%)and I left it for the weekend in the -20. However, I can see that the salt has precipitated to the bottom forming a lot of white precipitates! (obviously it is not DNA) and I am worry that I have lost my DNA sample as it is really precious from a colon biopsy. Could you tell me if there is anything I can do.
I have a question. In our PCR standardisation we used ethanol precipitation to reduce the concentration of non specific product. We stored the PCR product and 100% ethanol mix overnight at -20. Could you tell me if this will reduce the concentration of non specific product or do you suggest any changes? Thank you
er… as mentioned, adding salt (natrium acetate)is to neutralize the charge of the sugar phosphate backbond. May i know
1) If total quatity of aqueous solution i had is 800µl. How much of salt and ethanol(95%) i need to add in?
(normal procedure is 0.1 volume of salt with 2.5-3X volume of 95% alcohol, right?)
2) If i had added 1 volume of salt accidentally and just added 1volume of alcohol? what will happened?
3)Any countermeasurement for that situation?
What would happen if you added sodium acetate to a final concentration greater than 0.3 M? Would that affect my RNA even though I perform a wash.
The story behind the question”
I am doing a phenol chloform extraction followed by ethanol precipiation.
I decided to increase my reaction volume by adding salt so i would not have to do this later in my ethanol precipiation. That way I increased the volume in which to do my phenol extraction.
I then added 100% ethanol and pelleted my RNA.
Hello Nick
Nice to read your description. Easy to know the concept.
Thank you very much
Pree
Two stupid questions:
1. How to prepare 70% ethanol for DNA or RNA purification?
2. How is it important that 100% and 70% ethanol must be cold during DNA or RNA purification?
Hello,
Your note is very clear and interesing.
I think Very impotrant is pH of Sodium acetate !
For prepare 1 litre of 70 graduate ethanol you have to mix 665g of EtOH 96% and 335g of H2O.