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
Great post – more please!
How phenol extraction works!
How CsCl centrifugation works!
How HPLC works!
Thanks for the suggestions – I’ll definitely write these articles in the future. Suggestions are always welcome!!
Thanks for this article. I have extracted DNA hundreds of times but wasn’t 100% sure how and why it worked. Thanks for the explanation!
I’m a little bit freaked out by the timing of this post since I was just thinking about this very topic yesterday… Oh well, perfect timing. Thank you very much.
How does the ethanol wash the salt away?
Arag,
Thanks for your question.
The wash step removes the relatively small amount of salt that pellets with the DNA. This small amount of salt can dissolve in the 70% ethanol/30% water mix – mainly in the water part as the salt is far more soluble in water than in ethanol.
Is there optimal amount for Ethanol in precipitation? I heard people say more Ethanol you put in the solution, more precipitation you will get. But I always have to centrifuge SEVERAL times at 150000 rpm! Every time I centrifuge, I get more pellet out! I centrifuge 35min after freezing over night, then remove the ethanol to a new tube. After 5min in room temperature I centrifuge 30min again, then even more products can come out! What can be reason! I put 4.5 V Ethanol. Can that be the reason?
I am training a whole bunch of new people in my lab now and this little article just hit the spot. Thank you.
Very nice site you have. I’m going to subscribe to the RSS. Anyway, I still don’t understand why we need cold ethanol at all. Why not at RT?
Thanks!
@Ying
I;m pretty sure the optimum is 70% ethanol. As I understand it you need some water in there so that salts don’t precipitate with the DNA.
There are always losses associated with EtOH precipitation, but I think it is the still-solubilized DNA that was the reason for the losses.
I suppose that the longer you centrifuge the more you would get out, up to a point, but I don’t think the reason you are seeing this is the excess ethanol.
@Anne
Glad you like the site. The whole procedure is based upon making the DNA insoluble so it precipitates out of solution and can be pelleted.
The lower the temperature, the less soluble things are generally, so the low temperature helps to make the DNA insoluble.
is it possible to co-precipitate salt into the sample if we spin the sample at higher speed or for longer period?
Hi Cookie
I don’t think so. The salt will only precipitate if there is insufficient water for it to solubilise in. If it is in solution, no amount of centrifugation will pellet it.
cool site
Cookie,
Just do the wash in punctilious or 95% EtOH or skip the 70% rinse altogether. The salts aren’t soluble in 95% EtOH and will precipitate.
I worked with Jim Hartley & Jil Zeugin in the 80’s at BRL (now know as Invitrogen but really not the same). They published a study in 1982,which is still disputed by some cement heads, proving that the amount of time in the centrifuge is the primary determinant of yield and that cooling the Ethanol actually reduces yield and that incubation has no effect on yield.
So put your tube right into the ‘fuge after adding your RT EtOH and spin an extra 10 minutes (instead of having it sit on ice) and I promise your yield will increase.
i have been trying to find the Zeugin & Hartley reference, but since it was published in our trade journal (FOCUS)I can’t find it on the web.
Thanks for the explanation – very useful. Why is it that sometimes the DNA just won’t precipitate out at all? I had this problem today. I tried cooling and even adding glycogen – nothing!
dewi
Any sugestion how increase amount of DNA. Precipitation was done after reaction with BigDye 3.1. Fragment length 80 – 150 bp.
wonderful!
how many experienced people in a lab wouldn’t be able to explain it that well
thanx for the information,but i want to know if peg is having any effect on rna precipitation
What is the ‘dynamic range’ of precipitation in term of fragment length? That is, do small fragments and nucleotides precipitate as well?
shhigg,
The “dynamic range” of ethanol precipitation is in the range of 18-20 nucleotides, depending on their sequence. Over 20 bp and they will precipitate.
Free, non-bound nucleotides will not precipitate under standard conditions.
Smaller length oligos, RNA or DNA fragments are purified using one of the many commercially available “spin columns” or some other solid matrix, like biogel (per maniatis pp 11.34-11.37)
I have question. I have been isolating BACs in 96 well blocks for a while now and I do not get consistently good results. The results I hope to achieve are to have clear samples of DNA dissolved in water. However, I often get white junk in my well at the end that will not dissolve. The white junk appears after I transfer lysate from the wells (after I have added a cell lysis solution of NaOH and SDS then finally adding potassium acetate and leaving on ice for 20mins) into 300ul of isopropanol to precipitate the DNA. I sometimes let it precipitate at room temp for 20mins or sometimes at -80 for 15 (which is better?)before spinning it at 4000rpm for 30 minutes. The last step is washing with 70% etoh . I dont understand why there is white stuff in my wells that do not dissolve after I dry off the the ethanol and water. can you tell me which step could be affecting these results.
Thanks for good explaination.
I’ve got one question. Why do we sometime also expected to add Sodium EDTA together with Sodium Acetate for precipitation?
@Ali: It sounds like the white junk is the cell debris and the reason is a problem with the centrifugation step. 4000rpm is too slow to separate the supernatant from the white debris so with these centrifugation parameters you will always have some of the debris carry over into the supernatant. Centrifuging at 10,000 rpm+ for 15-30 minutes should help.
@Dana: Sounds to me like the sodium ions will help with the charge neutralisation of the phosphate backbone and the EDTA will chelate any available Mg2+, preventing nucleases from damaging the DNA after precipitation.
Hi,
I just heard that DNA will not precipitate if the Ethanol/isopropanol is added before the salt. Is this true? By accident I did this last week and I still retrieve much of my DNA. I do leave the tubes overnight in the -20C. So did I retreive my DNA or something else?
Thanks for your help
Hi Magy
The order in which the ethanol and salt are added will make no difference so it sounds like you got your DNA, although you’d be best to check on a spec and/or a gel to make sure!
Nick.
Thanks for this very nice website. It’s quite helpful.
1. About the “final conc” of the salt, does it mean the conc in the solution including ethanol (or before adding ethanol)? What would happen if the conc of salt is too high?
2. In some protocols, people use 2V of ethanol for DNA precipitation. What would be the optimal ratio?
In precipitation procedure,I got white cloudy soln after adding chiled ethanol?Why it happens?
Why do I need less isopropanol (1V) if compared to ethanol (3V) when precipitating DNA/RNA? Why is it not necessary to add salt when using isopropanol? How can I justify it in chemucal terms?
I am currently trying to run PCR on some samples that underwent EtOH precip. protocol over a year ago. The samples were stored at -70C and when i took them out of the freezer they were very cloudy. After doing PCR and running a gel I received product for my control, but did not receive product for any of the samples that were cloudy. I was considering running the EtOH precip. again, but have limited sample material. Any idea why these sample are cloudy and/or what can be done with them?
@CL: The final conc means the concentration after everything has been added, including the ethanol. 2 volumes of ethanol is added to give a final concentration of 70% ethanol. I believe this is optimal, but I am not 100% sure.
@Paula: Less isopropanol is a less polar solvent than ethanol, so you get the same effect as ethanol but with a lower concentration of solvent.
@Sarah and anand: The most likely reason for the white precipitate is contamination with protein. You could try cleaning up the solution with no loss of product using something like Stratagene’s Strataclean resin.
Thank you for this website. I just have one question: how does the 70% ethanol wash the pellet at the end?
@Yara. The salt is very soluble in water, so will easily dissolve in the water in the 70% ethanol
but why doesn’t the DNA dissolve as well?
[...] Re: DNA Nucleic Acid Precipitation There are some answers but no references in this blog post. [...]
why do v have to incubate at -80 after adding ethanol..generally temp is reduced to decrease the movement of molecules..wht is it doing here??
this is an awessssome site!!!!!!!!!!
Here is a suggestion to increase the recovery yield especially for very low amount: You can add linear acrylamide before adding Ethanol at a concentration of 5 or 10 µg/ml.
Other carrier as been used in the past such as glycogene. Acrylamide is better since no absorption is observed at 260 nm (which is not the case for most glycogene preps)
Thanks for the site it is very helpfull
Thanks for the explanation~
I have to extract RNA from skeletal muscle but the 260/230 ratios are not consistenly good (260/230 ~ 1.8-2.0 is good? for mine it’s ususlly 0.9-1.3). Is there a way to improve this?
Thanks! You’re explanation helped me a lot!
Cool site!
Thanks for the explanation. But what about ISOLATION OF YEAST RNA? What makes yeast an ideal source for RNA extraction?
@Yara. I should explain it the other way around : the DNA does not dissolve because it is poorly soluble in 70% ethanol but the salt is soluble enough in 70% ethanol to dissolve, so the DNA and salt are separated.
@Ritu. The idea is that for most solvents, including ethanol, the solubility (i.e. the amount of solute they can hold) decreases with temperature. So by lowering the temperature, you theoretically lower the amount of DNA that the ethanol can hold in solution. (see http://en.wikipedia.org/wiki/Solubility#Temperature). But according to Jim (see comment near the top of this list), ethanol precipitation works just as well at room temperature as -20/80.
@Cyril – Thanks – that is useful
@Ange – It might be the kit you are using. I find that the ratios are bad for certain kits and nothing seems to improve it. But if the RNA you have isolated is doing the job without any problem then there is no problem… just because the ratio is bad does not mean that the sample is no good, it is just one indicator of quality.
@Fe – I suppose yeast is only an ideal source for RNA extraction if you want to do something with yeast RNA. If you mean why is yeast used for commercial preps of RNA used for (e.g.) blocking the answer is probably that yeast is a relatively cheap source of RNA – economical to grow and relatively easy to break open and isolate the RNA.
Need help!!
Recently I tried to purify RNA (46 bases) with 20% denaturing PAGE. After finishing the gel running, I dyed the gel with EB and then cut the band of RNA under UV. Then I put the gel slice into elution buffer (0.5 M NH4OAC, 0.1M EDTA, 0.1% SDS) at room temperature over night and final volume is about six ml. After that, I did butonol extraction experiment and a final solution of 0.4 ml was obtained. 1 ml Ethonal was then added. The tube was buried in dry ice for two hours and much white precipitate was observed. I centrifugated the tube 30 min and got a large amount of precipitate and at the same time, the upper supernatant contains two layers. I removed the supernatant and used 70% ethonal (in DPFC water) to rinse the precipitate twice in order to remove the salt components. Then I dried the precipitate left and made a solution of about 0.4 ml with DPFC water. However, the OD measured is about 0.072 (the crude RNA used is 6 OD). It seems that there is no RNA in the final product.
What is the problem with my procedure?
How to purify RNA with length below 100 bases by native PAGE?
Thanks a lot!
i am exprience an unussual problem. ETOH precipitation after restriction with VSP1 resulted in an additional band that was not seen in a gel before the precipitation. The water used for 70% etoh preparation and the TE used to dissolve the DNA were checked for contamination and were found negative. Any idea someone? Can etoh triger an unspecific resriction?
Avi,
You don’t give enough information about the DNA you’re cutting, but I wouldn’t suspect your EtOH or water.
One possibility, especially if you’re doing a restriction digest of a plasmid, is that you’re seeing both the linear and supercoiled form of the plasmid.
Typically, “contamination” with non-specific nuclease will just give you a smear and not a distinct band on a gel.
Hi,
I’m having a similar probelm cleaning my plasmid digests. After digestion, I phenol:chloroform extract the DNA and then extract once more with chloroform to ensure removal of trace phenol. At this point, the digest looks the same as before. THE PROBLEM is after I ethanol precipitate, there is a ton of streaking and the larger bands are sometimes wholly absent. Am I seeing DNAse contamination?
M
Streaking maybe due to too vigorous pipetting or vortexing, especially if DNA is long. It can also be due to overdrying of DNA after precipitation.
After looking for quite some time I finally located two good articles about ethanol precipitation, one of them is the one Jim H. was talking about, they are available in archive on invitrogen website.
Hmmm, it seems I can’t post the links here I added them to wikipedia article on Ethanol Precipitation so just google it and look at the bottom of the page on wiki.
I also added links to them to wikipedia article on ethanol precipitation.
Zeugin JA, Hartley JL (1985). “Ethanol Precipitation of DNA”. Focus 7 (4): 1–2.
Crouse J, Amorese D (1987). “Ethanol Precipitation: Ammonium Acetate as an Alternative to Sodium Acetate”. Focus 9 (2): 3–5.
After doing ethanol precipition, I put my DNA in the -20 freezer so that I can run PCR the next day. Am I supposed to centrifuge my DNA sample again before I run the PCR? if so, for how long?
hey i want to know why only the dna containing part floureses after staining the gel with EtBr ? why not hte whole gel flourese ?wat change Etbr undergoes which makes it flourese
Hi,
I want to know about the required amount of salt to precipitate DNA. in my case, to precipitate DNA 4ug/50ul,
I added 3M sodium hydroxide 5.5ul and 7.5M ammonium acetate 1/3 Volume. After that, I added 2.5 Volume of EtOH.
But if add 7.5M ammonium acetate 50ul, is it ok to precipitate this DNA??? Please, answer to me.