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DNA Precipitation: Ethanol vs. Isopropanol

As a follow up to our article about ethanol precipitation of DNA and RNA, this article explains the differences between DNA precipitation in ethanol and isopropanol, helping you to figure out which method is the best choice for your experiment.

Requirements for Precipitation

First, let’s review the components we need to precipitate DNA or RNA with ethanol:

1. Salt to neutralize the charge on the nucleic acid backbone. This causes the DNA to become less hydrophilic and precipitate out of solution.

2. Ice to chill the sample. Lower temperatures promote the flocculation of the nucleic acids so they form larger complexes that pellet under the centrifugal forces of a microcentrifuge.

3. A nucleic acid concentration high enough to force the DNA out of solution (if the concentration is not high enough, you can add a carrier nucleic acid or glycogen to enhance the recovery).

4. A microcentrifuge to pellet the sample.

Isopropanol Vs. Ethanol: DNA Solubility

DNA is less soluble in isopropanol so it precipitates faster even at low concentrations. The downside however is that salt will also precipitate in isopropanol. With ethanol, the DNA needs to be at a higher concentration to flocculate but the salt tends to stay soluble, even at colder temperatures.

DNA precipitates in 35% isopropanol and 0.5 M salt. Using ethanol, the final concentration needs to be around 75% with 0.5 M salt. So for the typical precipitation protocol, isopropanol is added from between 0.7–1 volumes of sample and ethanol is added at 2-2.5 volumes of sample.

Choosing the Right Solvent: Sample Volume

If you are precipitating small volumes of DNA, and you can fit the required amount of solvent into the sample tube, then ice-cold ethanol is the preferred choice. You can chill it (in liquid nitrogen or at –80°C) to accelerate the precipitation without the risk of precipitating excess salt. Afterwards, you need to wash the pellet with 70% ethanol to remove any salt present.

Isopropanol is useful for large sample volumes e.g. the eluate you get after using a large volume plasmid kit. Because less isopropanol is needed for precipitation, you can often fit your sample and the solvent in one 15 ml tube. However, because salts are generally less soluble in isopropanol than in ethanol, they tend to to co-precipitate with DNA. To minimize the likelihood of salt precipitation, isopropanol precipitation is best at room temperature with short incubation times. Once you recover the DNA or RNA pellet from the isopropanol, wash it with cold 70% ethanol to remove excess salt and to exchange the isopropanol for ethanol. It is ok to chill the isopropanol-precipitated sample, if you are sure the sample doesn’t contain a lot of salt.

Because DNA is less soluble in isopropanol, isopropanol allows precipitation of larger species and lower concentrations of nucleic acids than ethanol, especially if you incubate at low temperatures for long periods of time. If you do this, just remember to wash the pellet several times in 70% ethanol after pelleting, to reduce the amount of salt you carry over.

In Short – Ethanol or Isopropanol?

Use Ethanol If:

  1. You have the space to fit two volumes of ethanol to sample in your tube.
  2. The sample needs to be stored for a long period of time and will be chilled.
  3. You need to precipitate very small DNA fragments.

Use Isopropanol If:

  1. Your sample volume is large and you can only fit 1 volume of solvent to your tube.
  2. You need large molecular weight species.
  3. The DNA concentration in your sample is low.
  4. You are in a hurry and want to accelerate the precipitation of nucleic acids at room temperature.

Handy Tips

  • Ethanol precipitation:
    • Add 2 volumes of ethanol to the sample and freeze at –20°C for at least 1 hour or overnight for best results.
    • Centrifuge the sample at full speed for 20 minutes to collect all material.
    • Wash with 70% ethanol, then centrifuge for 10–15 minutes to pellet the DNA.
    • Remember to mark the side of the tube where the pellet is expected to be and don’t let it out of your sight when decanting the ethanol!
  • Isopropanol precipitation:
    • Avoid cold temperatures because of the excess salt precipitation that can occur.
    • To increase the yields precipitated, incubating the sample mixture at room temperature for longer periods rather than chilling the sample.
    • When the DNA is pelleted, the pellet is sometimes more difficult to see compared to the ethanol pellet. It can be clear and glassy. Make sure, again, to note the side of the tube where the pellet should be. Look for it before decanting the isopropanol and 70% ethanol wash.
    • After washing with ethanol, the pellet becomes visible and white. Make sure it doesn’t slip off the side of the tube wall before decanting the supernatant. Allow the tube to drain upside down for a few minutes, let it air-dry or use a centrifugal evaporator (5 minutes is enough) and then resuspend in buffer.
  • Finally, for dry DNA pellets, heating the sample in buffer at 50–60°C will help the DNA dissolve faster and won’t damage the DNA. Heating is also suitable for RNA, in a water bath at temperatures not exceeding 42°C. Over dried DNA and RNA will take longer to dissolve so make sure not to evaporate for too long.

So now you know the difference between ethanol and isopropanol precipitation, and when to use each method. Good luck with your DNA precipitations!

Originally published on December 10, 2009. Updated and republished in May 2017.

60 Comments

  1. Laura on December 12, 2019 at 10:13 pm

    Hi in my script it is nowhere mentioned that we are supposed to use salt. Does it still work without adding salt or is it a mistake in the script? And also we are still supposed to wash the pellet with ethanol but to me that doesn’t make any sense if we didn’t add salt. Could you please help me out?

  2. Claire on February 2, 2018 at 10:57 am

    Thanks for this clear explanation. It helps a lot the students who discover the beautiful world of molecular biology !

  3. Mirza on September 20, 2017 at 12:26 am

    Hi,
    thanks for all nice information,
    I extracted DNA using Ctab and at last I diluted it with ddh2o for checking the quality of DNA using agarose. I’m having some problems with re-pellet my DNA for shipping, please advise the protocol for re-pelleting my DNA..

    Thanks from Indonesia

  4. yaqub on August 14, 2017 at 9:33 am

    I want to do sequencing for from my DNA which is from PCR, and I isolate PCR product by agarose gel, and electro elution, my target size is 450bp around. But I always lose my DNA at Ethanol precipitation step. I add 3M sodium acetate, glycogen, and 100% ethanol, -20degree 10min, 4degree 15000 centrifuge, I saw my pellet, and wash by 70% ethanol, pellet still there, solubilize it by DW, after a check on the gel couldn’t see any band. which step has a problem?

  5. marzieh on June 12, 2017 at 6:25 am

    hi
    i wrongly used 50 sodium acetate 3M for 50 ul solution in Ethanol Precipitation of DNA. nowely what should do i?

    • Dr Amanda Welch on June 13, 2017 at 7:23 pm

      Did you use too much sodium acetate? If so, then you can just increase the volumes proportionally for the rest of the precipitation. Just keep in mind that your recovery might be a bit lower.

  6. Bruce on June 9, 2017 at 12:53 pm

    Thanks for this useful explanation.

    I would be grateful if you are able to explain how this protocol works:
    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3441248/
    It seems to do the opposite of everything: it dilutes the salt which should be what makes the dna drop out of solution, it doesn’t use ethanol or isopropanol and it incubates at 60degrees rather than chills during precipitation.

    Many thanks

    • Dr Amanda Welch on June 13, 2017 at 7:37 pm

      Skimming over this, it looks like they authors used magnetic beads to do the precipitation—so that’s why there’s no alcohol involved. The heating looks like it’s to denature the proteins (filling the same role as Proteinase K does during DNA extractions).

  7. Dave on June 3, 2017 at 2:56 am

    Hi there, I am extracting from tissues and using isopropanol, but after adding the 99% isp the usual big white fluffy DNA strands isnt there anymore, needed to centrifuge at high speed to get the DNA pellet, which after specing turns out to be 200ug, which is lots, my question is why dont i see the strands of DNA PPT out of solution anymore?! Did this in a batch of six and none of them ppted out with a strand, just turned cloudy and needed centrifugation….Are my solutions off? Using Qiagen puregene kit, basically, there is lysis buffer, protein ppy soln, and isp…..Thats it.

  8. Sara W on May 30, 2017 at 10:52 pm

    I’m looking into doing an ethanol precipitation to purify my DNA samples and hopefully improve my 260/230 ratios, which are low. I did have a question about freezing the sample overnight once the ethanol had been added. I froze my tissues after sampling and am concerned that another freeze/thaw cycle with the ethanol precipitation will compromise DNA integrity. Any ideas on this? Thanks!

    • Dave on June 3, 2017 at 2:58 am

      Just use a zymo kit to concentrate and cleans dna in about 15min, works super well, we use it to clean genomic dna samples that just has lots of contaminants in them by nature….Ie formalin fixed, amniotic fluids etc…

    • Phuong Nguyen on June 23, 2017 at 1:44 pm

      In my experience, preserve DNA as pellet under ethanol is fine, if not the best method to store DNA for a long time.
      I always having problem with RE digestion of plasmid DNA that are more than 1 week old, longer storage may lead to DNA damage as I see an additional band of nick DNA on agarose gel (no, we didn’t have the nick band earlier because we prep plasmid to obtain supercoiled DNA only).
      Recently, I found out that, I could make a large quantity of plasmid DNA, purify it, precipitate them with alcohol and salt of choice, aliquot into 10-20 epi before place them in -80*C like forever. We just need to centrifuge the aliquot and wash it with 70% alcohol as usual, the DNA are always at its best, supercoiled, and digestion product was sharp like commercial DNA ladder.
      It may come in handy if you do a lot of cloning, no need for DNA prep every week.

  9. Romelio on May 18, 2017 at 12:39 am

    Hi, for the frist precipitation of DNA
    do you use etanol 100% or 70% + salts ej sodium acetate? then wash with etanol al 70 %

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