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.
[...] A: The answer to this question is: I don’t know exactly, but ammonium or sephadex might help you. The size of the DNA that can be removed by ethanol precipitation using various salts and ethanol vs. isopropanol has never been critically analyzed down to the detail you desire (as far as I could ascertain using my incredible google research skills). An overview of the different salts used for ethanol precipitation and how to choose has been described before by Nick in this article. [...]
Very nice, the explanation is good, but leaves things a little ambiguous… for us students – could you add a thermodynamic point of view? It would make everything so much easier to understand…
For some special purpose, I run my DNA sample on a regular agarose gel, then cut the bands. I use 4.7M (final conc) NaI and 42C incubation to melt the agarose gel block with DNA. At this step, can (and how) I use ethanol to precipitate the DNA? Since there are already plenty salts (NaI at 4.7 M), do I still need to add NaOac as usual? If I put this DNA-gel-ethanol in low temperature, can the gel re-solidify and then precipitate with DNA?
Thanks!
Nothing to add, but great info!
Sir you explain all things right no doubt,but i have a query that why we use only Chilled(Ethanol or Isopropanol)i.e whats the need for chilling?
I am very thankful to you if you help me out in this.
Ohh i get the ans as Jason asked the same question to You.
Thanx alot sir,I am very thankful to you.
Plz keep it up…
hi nick,
i am isolating RNA from a plant sample. i use isoprapanol for precipitation, But everytime i got a lot of contamination of polyphenols with RNA. Can u help me to get pure RNA??
Hi Nick,
I would like to ask you about dna rna precipitation: why dna stays at organic phase at acid ph and rna at aqueous phase?
thanks
Every time I do an ethanol precipitation of DNA, I find white flakey precipitate in my sample after resuspending in either ddH2O or Tris pH 8.5. This happens regardless of how well I try to wash the pellet with 70% room temperature ethanol. Does this happen to anyone else? Is it just left over salt that is no longer soluble for whatever reason? Does it affect downstream steps? It doesn’t appear to affect transfection, but I am wondering about enzymatic reactions where the salt concentrations matter (e.g. ligation).
Hi John,
Are you using a silica column method for purification? Or an anion-exchange column (such as qiagen plasmid kits)?
Sometimes the resin leaks through into the final samples. If this is not affecting any results, you can spin it out and transfer the DNA to a new tube.
Best,
Suzanne
Hi Suzanne,
Thanks for your reply. I do the ethanol precipitation after extracting the DNA from agarose with a Qiagen kit. I always like to do an ethanol precipitation after gel purification to remove any excess salts or left over agarose. I find this helps with downstream ligation. I only see the white precipitate after the ethanol precipitation, not after eluting the DNA off the column. I suppose its possible that the ethanol precipitation knocks the column “leakage” out of solution and it remains insoluble afterwards. Is this plausible?
Hi John,
It is possible. I recall hearing this from other users when I worked at Qiagen (that after ethanol ppt. it appears). If you give them a call and ask about it, they might be able to confirm that it is indeed just a little resin and to remove it, or if it is something else.
If the DNA is performing fine in reactions, it is probably just resin.
Best,
Suzanne
What is the physical different between precipitation of RNA from precipitation of DNA using ethanol
Hello Sir,
I wanted to ask you that why it is so that some DNA samples immediately precipitates out easily as soon as chilled ethyl alcohol is added whereas, the others requires storage for about 15-20 minutes at -20C and also I wanted to know that what is the role of Ethyl alcohol in DNA precipitation. I will be very thankful to you if you help me out in this.
I am trying to precipitate DNA using the commonly used ETOH method.However, I can see that the salt has precipitated to the bottom forming a lot of white precipitates!Could you tell me if there is anything I can do.
i used ethanol precipitation. but at the lots of salt precipitated with DNA. is there any way to clean up the salt because the i have extracted the DNA from gastric biopsies.
[...] The Basics: How Ethanol Precipitation of DNA and RNA Works [...]
Hey there,
would you tell me the soucre of your statement, that NaCl should be used when SDS is present in the preparation?
Greetz, Chris (Karlsruhe Institut of Technology KIT, Germany)
http://www.kit.edu/
Hi Chris,
Maniatis is one source and if you don’t have that (it’s old) then check the new Sambrook and Russel Molecular Cloning Manuals.
The appendix has a section on ethanol precipitation.
Suzanne
(also- personal experience- I tried precipitating DNA that had SDS in the lysis with NaAcetate vs NaCl and without NaCl it was a mess.)
THX a lot…I will check the literature. Ok, I’ll stay with my NaCl
Greezt, Chris
Hey
I have a question, do the PCR purification kits help in desalting DNA? Im doing something where Im eluting ssDNA with a buffer containing Urea…can i use these kits to get rid of the Urea? Or should i do a std ethanol precipitation? will that help get rid of the Urea? Im trying to avoid dialysis..
Hi,
I have extracted genomic DNA from my samples and quantified by using UV absorbance and fluorescence-picogreen to check the sensitivity of the quantitation method. The outcome was, DNA yield quantified by picogreen are almost 3 times higher than the yield in absorbance. The ratios of A260/280 are within the range (1.6-2.2) but the ratios for A260/230 are very very low in range 0.28-0.5. I used salt out method to extract the DNA. Do you think the reason because it is high salt in my DNA or because I didn’t wait to dry the pellet after DNA precipitation with 100% EtOH, followed by 70% EtOH wash step before adding the TE buffer?
Therefore, is it possible that I still have ETOH in my DNA which makes the DNA difficult to dissolve completely and only when quantified by picogreen which bind specifically with dsDNA it gives high yield?
From what I have read in previous paper, DNA yield by absorbance are higher compared to picogreen.
Please help me to understand this situation.
Thans a lot. Sorry for very long post…
moderator-edited post because of spelling errors.
hello there… i have problem with my DNA precipitation. i’m isolating DNA from NPC cell line bdw. it really take me longer time to pellet the DNA after the addition of absolute ethanol. what might be the problems?? i need to do around 4 to 5 times of centrifugation at high speed for 10 mins to pellet the DNA. usually my dna will be sheared or degraded during this procedure
hello ,Your explenation is so intresting
many many thanks
but what is the role of low tempreture in precipitation?
Hi Nick
I have a qouestion. I am doing HRM analysis for SNP genotyping. Salt concentration of DNA samples has high effect in this analysis. the DNA pellets are dilouted in TE. Would you please help me how to precipitate salt from my Samples without extracting DNA again?
Many thanks in advance
Quick question – if the purpose of the salt is to neutralize the backbone charge, why is the final concentration of the different salts used so disparate?
Also, in trying to check calcs, if one uses 0.1X NaOAc (stock @ 5M)and 2-2X EtOH, I don’t calculate a final concentration of 0.3M…??? Example, resuspend sample in 1 ml, add 0.1 ml 5M NaOAc, add 2 – 2.5 ml EtOH. I get (0.1 ml)(5M) = x (3.1 ml), x = 0.16 (or if using 2.5 ml EtOH, x = 0.14.
What am I missing?
Sorry for being an idiot, I figured out the calcs moments after posting, it’s 0.1 volume of a 3M solution of 3M NaOAc, so the 0.3M “final” concentration is referring to the concentration of the salt in solution PRIOR to addition of EtOH.
Still curious about the different final salt concentrations though!