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DNA Precipitation: Ethanol vs. Isopropanol

Image of raindrop on a leaf to represent DNA precipitation

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 DNA 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. [1] 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 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 into 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 of DNA:
    • 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 of DNA:
    • 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!

 

References

  1. Green MR, Sambrook J. Precipitation of DNA with Isopropanol. Cold Spring Harb Protoc. 2017(8):pdb.prot093385.

Originally published on December 10, 2009. Updated and republished in August 2021.

60 Comments

  1. ADAN CHAVEZ on April 19, 2017 at 7:19 pm

    Ethanol 70% must be cold or at ambient temperature?. I am not sure.



  2. evi on April 6, 2017 at 8:32 am

    Hello, i’m using the C-tab protocol for DNA extraction and i’m wondering how long can i leave the sample with isopropanol in -20 °C. The protocol says overnight but can i leave it for a weekend or long ?
    Thank you !



    • Dr Amanda Welch on April 6, 2017 at 1:29 pm

      I’ve left it for a couple of weeks and had good recovery (tl;dr mix up between me and the lab tech about who was doing what). So, I think you’re ok with a long weekend. That being said, if it was a precious sample, then I’d follow the protocol.



  3. James Hinge on February 27, 2017 at 10:28 am

    when doing a kids experiment on dna by extracting dna from a banana or rasberry what type of alcohol is normally used??? i am a new science teacher for the after school in my school..



    • Marie on March 30, 2017 at 9:19 am

      Hi James Hinge

      With the caveat, that I am not a teacher, I have just done this experiment for fun at home, I would say that the best would be 96% ethanol if you can get it, but I have also gotten it to work by using vodka with a high alcohol percentage 🙂 Probably will not give you as good a yield, though, but I just wanted to show the solution becoming clouded, and that was possible with Vodka (probably 40%/80 proof). Depends what you want with it, so may take some experimentation, but generally I would say 70-96% would give you the best results.

      Have fun!

      Marie



    • Dr Rebecca Tirabassi on April 1, 2017 at 12:19 pm

      I’ve done this for my kids’ classes. We used strawberries, though. The higher the percentage alcohol, the better. If you can order 100% isopropanol it will work better. Otherwise you can use the stuff you find at a drug store. Just make sure it is ice cold. If you need a protocol, let me know. [email protected]



    • Phuong Nguyen on June 23, 2017 at 1:55 pm

      I have tested both isopropanol 100% and alcohol 96% for this experiment before. Base on my observation, Isopropanol seem to work better than alcohol, but they did not make significant difference except that it took less time to see the precipitation occurred.
      And you do not need to freeze the alcohol prior to the experiment, you still can get the same fluffy precipitate (even though I was not sure it WAS DNA, too lazy to run it on gel, kid don’t mind if it is anyways).



  4. Josh li on February 20, 2017 at 1:44 am

    I extracted DNA from plant, M. truncatula using CTAB method. And my downstream application afterward is RTqPCR. I have good OD results too. I have fine band in gel after standard PCR. However, no signal is seen in Real time. I use DNA amount ranging from 100ng to 1ng with primer 100nM. I changed DNA and primer concentrations many times. some sample show amplification with only 1ng DNA. Facing real hard time to fix this issue. I would appreciate anyone’s idea in here.



    • Dr Amanda Welch on February 20, 2017 at 3:51 pm

      Have you done a control reaction with your RTqPCR? That would give you an idea if something is wrong with your polymerase or other reaction component.



  5. TB on February 3, 2017 at 8:25 pm

    I am wondering if there is a difference between -20C incubation v -70C incubation (other than differences in times)? Is one a more efficient method to use ethanol precipitation for DNA concentration?



    • Dr Amanda Welch on February 4, 2017 at 1:02 pm

      As far as I know, it’s primarily a difference in times. In my experience, I did not get any difference in yield between the two different temperatures. However, if you were dealing with rare samples, then I’d be a bit more careful. 🙂



  6. Jose A. Quinteros on September 12, 2016 at 9:57 am

    I have a question. I’m amplifying plasmid and DNA using PCR and the size of my amplicons range from 5.7 Kb to 7.2 Kb. I need to concentrate these amplicons because I’m planning to use them in a Gibson assembly reaction. Would the ethanol precipitation cause any damage to these amplicons (considering they are relatively big).
    Thanks a lot for your help!



    • C on October 19, 2016 at 9:52 am

      No.



  7. yael on August 10, 2016 at 8:08 am

    I understood that the centrifugation needed to be done at 4C.
    But what will happen if i did Isopropanol centrifugation for 1Hr, but the centrifuge was started at 31C and than cooled down to 4C during the centrifugation (with the sample). So it took it about 30min to cool down to 4C with the sample inside and than the remaining 30min the centrifure worked at 4C.
    Can it effect the DNA quality or just the quantity ?



  8. Stephanie on January 28, 2016 at 4:19 pm

    HI,

    I’ve a question, why do you use ethanol at -20˚c? Do you get better results with cooled ethanol than with room temperature?



    • Dr Amanda Welch on January 28, 2016 at 6:44 pm

      Hi Stephanie,

      Yep. It helps speed up the precipitation.



      • Stephanie on January 29, 2016 at 2:21 pm

        Thanks for the reply.
        How does it exactly speed up the precipitation.



        • Serge on March 31, 2016 at 1:04 pm

          As a general rule in physics, all molecules are less soluble at lower temperature…



      • Guillaume on June 15, 2016 at 8:42 am

        But if you cool the sample after pouring ethanol, you don’t need ethanol to be cold in the first place, when you use it, right?



        • Dr Amanda Welch on June 15, 2016 at 3:39 pm

          I’ve done it without cooling the ethanol first (and then sticking it all at -80C), but you do get better results with pre-chilled ethanol.



  9. Gen on November 19, 2015 at 12:49 pm

    Hi,

    Thanks so much for this useful post. I have a query regarding gDNA extraction from plants using silica/magnetic bead method for high throughput liquid handlers.

    Many of the plants were are working on have a high polysaccharide and polyphenolic content, and we are experiencing poor 260/230 ratios, likely due to carbohydrate carry-over. We have recently tested a high salt CTAB buffer with DTT and PVP, followed by isopropanol precipitation at RT, two 70% ethanol washes, and a final H2O wash (without agitation) to clean out the salts. We are still getting poor 260/230 ratios (1 or less). I would like to know if:
    1) Ethanol precipitation would reduce salt contamination (providing their is sufficient space in deep well plates), and sufficiently improve the 260/230 ratios?
    2) If it is better to avoid a high salt CTAB lysis buffer and opt for other reagents to remove the carbohydrates? If so, what reagents are recommended?

    Downstream processing of samples is for NGS prep, some of which involves enzyme digests. We have found that unless our samples exhibit near perfect 260/280 AND 260/230 ratios, digestion is problematic/incomplete.

    Any advice would be greatly appreciated!



  10. Daniel Damián Hernández De Castilla on April 19, 2015 at 2:29 pm

    Hi.
    First of all, thanks for this information, it is really useful.
    I have troubles with 230-260 ratio, usually I get values close to 1 or lower, instead of 2, but I can not figure out what I am doing wrong. I use Trizol and Quiazol for RNA isolation from cell lines.



    • Eduardo on December 9, 2015 at 1:55 pm

      Recall that Trizol is typically composed of GITC (guanidium Isothiocyanate) and phenol. The later absorbs at both 270nm and 230nm, so if you have carry-over contamination that will cause your purity to drop. GITC absorbs at 260nm,. similar to nucleic acids so beware of this contaminant as well.



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