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DNA Precipitation: Ethanol vs. Isopropanol

Since our most popular article of all time (“The Basics: How Ethanol Precipitation of DNA and RNA Works”) was published, many of our readers have asked us to further explain the difference between precipitating DNA with ethanol vs. isopropanol and which is the better choice. So today, I’ll meet the challenge and discuss the pros and cons of ethanol vs. isopropanol.

Requirements for Precipitation

First, let’s review what we know about what is needed for precipitation of DNA or RNA with ethanol:

1. Salt to neutralize the charge on the nucleic acid backbone, causing the DNA to become less hydrophilic and fall out of solution.

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

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

4. Centrifugation to pellet the sample

Isopropanol Vs Ethanol: DNA Solubility

DNA is less soluble in isopropanol so it will fall out of solution faster and at a lower concentration, but the downside is that the salt will too. With ethanol, the DNA needs to be at a higher concentration to flocculate but the salt tends to stay soluble, even at cold temperatures.

DNA falls out of solution 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: Volume of Sample

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 (some people use liquid nitrogen or –80°C to accelerate the precipitation) and precipitate more DNA without the salt contamination that would occur from chilling isopropanol. Afterwards you need to wash the pellet with 70% ethanol to remove salt.

Isopropanol use useful for precipitations where you have a large sample volume  (e.g. the eluate you get after using a Qiagen plasmid Maxi Kit) because less solvent is needed, so you can fit the whole lot in the (15 ml) tube. But because salts are generally less soluble in isopropanol than in ethanol, they have more of a tendancy to co-precipitate with the DNA. So to lessen the chances of salt precipitation, isopropanol precipitations are carried our at room temperature with minimal incubation times.   Once the DNA or RNA pellet is recovered from the isopropanol, you’ll want to wash it with cold 70% ethanol to remove excess salt and to exchange the isopropanol with the more volatile ethanol. It is ok to chill the isopropanol precipitated sample, if you are sure that it is not excessively salty.

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 it cold and long. 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.

When to Use Ethanol or Isopropanol

Use ethanol if:

  1. You have room 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 pieces of DNA or you have a very low concentration of sample so you want to chill it longer and colder.

Use isopropanol if:

  1. You have limited in space in your tube and can fit only 1 volume of sample.
  2. You need large molecular weight species because incubation at room temperature for short periods of time will not be conducive to precipitating small species of nucleic acid.
  3. You are in a hurry and want to accelerate the precipitation of nucleic acids at room temperature

What do I prefer? I use ethanol over isopropanol for most cases, but will use isopropanol if I need to make everything fit in one tube. My preferred protocol is 2 volumes of ethanol and freeze at –20°C for at least an hour or overnight for best results. I centrifuge the sample at full speed for 20 minutes to make sure I get everything down. I always wash with 70% ethanol and then centrifuge for 10–15 minutes and keep my eye on the pellet when I decant everything. You need to note or 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!

If I use isopropanol, I avoid cold temperatures because of the excess salt that usually comes down with it. If I want to increase the yields precipitated, I prefer to leave it incubating at room temperature longer vs. 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. I always 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 and then air dry or speed vac dry (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. For RNA, heating can be used too (in water) at temps around 42°C. Overdried DNA and RNA will take longer to dissolve so make sure not to speed vac for too long.

So now you know the difference between ethanol and isopropanol and when to use which. If you have any questions, or anything to add, please drop a comment in below.

Originally published on December 10, 2009. Updated and republished on June 23, 2015.

58 Comments

  1. 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

  2. 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?

  3. 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.

  4. 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).

  5. 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.

  6. 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.

  7. 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 %

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

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

  9. 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.

  10. 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).

  11. 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.

  12. 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. 🙂

  13. 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.

  14. 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 ?

  15. 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.

  16. 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!

  17. 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.

  18. Tiago Bruno on February 19, 2014 at 11:51 pm

    Hello everybody. Thanks for the nice post about the differences about preciptating DNA and RNa in isopropanol and ethanol. I have a question about the cloud that appers after isopropanol addition in rna extractio. That is rna? And the strange fillaments changing the light refraction? Another useful help i would like to receive from you guys is why sometimes my rna do not preciptate in the bottom after centrifugation. It is so frustating. I make extractions from heart of mice and i should get at least 100-300 ug, but iam only getting 20-60. Thnx very much.

    • Suzanne Kennedy on February 24, 2014 at 6:24 pm

      Hi Tiago,
      Thanks for your question. I would say, yes, exactly, if you see filaments falling out of solution, that should be the nucleic acids. When that happens the yields are high. Often times you do not see it like that but I will sometimes see opaqueness forming and then it goes away.
      From heart muscle you should not be getting other contaminants such as polysaccharides forming and causing precipitation. What method are you using for extraction of RNA?
      Heart is a difficult sample type because it is a fibrous tissue. we have a post coming up very soon- this week on fibrous tissues and RNA. How much tissue do you start with? 100-300 ug of RNA from heart would have to be a very large starting amount. Tell us more about the method you use for RNA isolation and we can figure out where it is going.
      When you centrifuge to pellet the RNA, spin for 20 -30 minutes at full speed (16,000 x g or higher). What time and speed are you using?
      Best,
      Suzanne

  19. user-58341 on May 29, 2013 at 8:28 am

    Hi Suzanne,
    I am also facing some problems with BAC DNA isolation as the quality is not very high. I am using isopropanol at RT for DNA precipitation. What should i do to get pure DNA for fingerprinting.
    Thanks,

    Vinod

  20. Heather on January 9, 2013 at 12:02 am

    This is really useful information, thanks! My lab does not have temperature-controlled centrifuges though, and they do heat up the samples quite a lot after just 10 min at full speed. I wonder if the benefits of a 20 or 30 min centrifugation might be counteracted by the samples being heated? Is some of the DNA likely to resuspend?

  21. ask on September 14, 2012 at 6:11 pm

    thanks!

    • Adam S on January 4, 2013 at 6:58 pm

      If DNA falls out of solution at around 75%, why is it that we only add 66% final (2 volumes)?

  22. melody on September 1, 2011 at 5:53 pm

    Hi! How long will it take to heat the sample in buffer at 50-60C? (for dry dna pellets)

  23. Jorge M on October 30, 2010 at 7:14 pm

    Hi Suzanne,

    Nice blog.  I am having some trouble with purifying viral dsDNA.  Is there any significant different in solubility between this kind of DNA an mammalian?
    For example, last attempt I saw a lot of “DNA” after adding ethanol (+1/10 NaAcetate).  I centrifuged it for one hour, and the pellet was tiny.  I centrifuged further for 4 extra hours, and I got much more DNA.
    Comments will be appreciated.
  24. Nikolai Nikolai on October 11, 2010 at 12:22 pm

    Hi Suzanne,
    I’ve been working with RNA for a couple of months now and couldn’t seem to make a single protocol fit for the isolation of RNA of different plant samples. The method which uses LiCl precipitation would give me a good curve (and a good 260/280 ratio) with max. abosorbance at 260 nm for one plant sample. Other samples give out a peak at 265 – 269 nm, and still others would have no peak at all. Some of the pellets I get would be brown colored.. I usually use 100-300 mg of homogenized leaves as my starting material…I tried using RNA isolation kit based on the Chomczynski and Sacchi protocol but this gives me a constant peak at 269nm with poor 260/280 ratio for my samples.
    Do you have any suggestions for this results? Thanks. 🙂

  25. Ka on February 26, 2010 at 12:32 pm

    Hi there,

    I have a basic doubt. After transcribing cDNA from total RNA with Omniscript kit Qiagen)to perform real time PCR, do I need to precipitate it? If I do, will the pellet also contain RNA fragments and DNTPs, that were left from the RT reaction, together with the cDNA? If that is true, the quantification of cDNA by Nanodrop is superestimated, right?

    Thank you for the tips and help.
    Best wishes from Brazil,
    Ka.

    • Suzanne on February 27, 2010 at 7:08 am

      You don’t need to precipitate it after RT. You should go direct into the PCR step and use not more than 10% of the reaction volume with the RT reaction. The only reason to clean up the RT reaction would be to quantitate it first. If you simply precipitate the RT reaction without any clean up to remove protein, you will co-precipitate some of the reactants you don’t want. The easiest way to clean up the RT is to use a commercial PCR clean up kit or to use phenol-chloroform first and then precipitate.
      I don’t know if the dNTPs and RNA fragments would co-isolate with the cDNA during the precipitation but I would think that if you centrifuge it long and hard enough, you will get some of it coming down with your cDNA and if there is enough nucleic acid present, the small stuff will co-precipitate with the large stuff and they’ll stick together and all pellet together.
      So better to remove it before precipitation to be sure.

  26. DK on January 24, 2010 at 10:56 pm

    Cooling during alcohol precipitation of DNA does one thing very reliably though:

    It makes your DNA dirtier. With cooling, one ends up with more protein in the pellet.

  27. Suzanne on January 15, 2010 at 4:41 am

    Hi D,
    I have heard this from others before but never experienced it. Are you doing a phenol extraction and maybe carried over some of the organic phase into the aqueous? Might you be taking the wrong phase of the extraction (it should be the upper and not the lower)?
    Tell us more about what you are doing?

    Thanks,
    Suzanne

  28. D on January 13, 2010 at 10:43 pm

    Hi,
    I’ve been having trouble with precipitating DNA. When I try to the isopropanol precipitation, I end up with a separation of layers. One organic (oily) phase and one aqueous phase. No amount of mixing will give me a proper DNA pellet.

    Have you ever seen this before?

  29. Suzanne on January 10, 2010 at 12:49 am

    Hi Dan,
    I am not familiar with any studies looking at size but the current data from the papers Jim mentioned say that longer centrifugation is important for getting high recovery. Although the data is not strong, he suggests room temperature centrifugation so the DNA has an easier time moving through the liquid.
    I still will use cold incubation because it makes sense that precipitation or flocculation will be enhanced in cold.

    To purify a 30 bp nucleotide, I would use something like a sephadex resin instead of precipitation, if you can.

  30. Dan on December 31, 2009 at 12:24 am

    Great article–but one other thing I’ve been looking for is a discussion of the fragment sizes brought down by different precipitation methods. For instance, if you have a 30 bp oligonucleotide you wish to purify, is one method better than another? What are the lower fragment size limits of each method? And while we are at it, any advice concerning perhaps concentrating ds versus ss DNA? Any thoughts? Anybody? Bueller?

  31. Suzanne on December 16, 2009 at 4:01 pm

    Hi Marc,
    If the plasmids are low copy, you can try a protocol used for large constructs such as P1 clones and BACs where you start with a much larger volume, precipitate the DNA after the solution 3 step to condense it down, and then resuspend in the buffer for anion-exchange in a volume that fits in one load. I can direct you to a protocol if you need.

    Make sure you are not over growing the plasmid and you can avoid excessive carbohydrate in the final DNA. The E.coli strain has an impact on this too. You might want to change cells.

    Sometimes plasmids grow poorly in bacteria because of the insert. There are specialty competent cells you can try that help with toxic clones or unstable plasmids.

    Last- have you tried centrifuging at 4C vs. -20C? That article Jim posted said that centrifuging too cold can reduce yields because it becomes more difficult for the DNA to move through the viscous liquid. RT or 4C might be better.

    Best,
    Suzanne

  32. Marc on December 14, 2009 at 5:11 pm

    I for one have no other choice but to centrifuge precipitated DNA (maxiprep, isopropanol, total volume ~25ml) at less than 5000g, so I let the centrifuge run for at least 1 hour, 20°C, and usually get good amounts of DNA…except for some plasmids.
    Actually I wish there were some tutorial on maxiprep optimization in this website. I have tried the PDM medium as suggested here: https://bitesizebio.com/2008/04/28/pimp-your-plasmid-growth-medium/
    It allows for more bacteria to grow (per ml), and thus yield to more DNA…but also more protein, RNAs…
    I am working in the lentiviral vector field and have noticed that low maxiprep yields (<400ug) usually lead to poor transfection (because of the level of “contamination” I assume), so I am looking for a way to get high amount of highly pure DNA…
    It is probably not the right place to ask for this, but if you have any hint, I’d be glad to hear it.

  33. John Mackay on December 13, 2009 at 8:03 pm

    There was also a great paper in methods of enzymology (late 80’s I think?) that discussed the different salts used. From memory it also described that cold temp wasn’t required and centrifuge time (esp for NH4Ac – 30 minutes) was the issue.

    I’m interested to know what centrifuge times most people use for their precipitations? Many protocols are just 10 minutes but habit (ha!) usually sees mine at 15 or 20 mins (except for 30 mins for NH4Ac)

  34. Jim H on December 12, 2009 at 3:04 pm

    Suzanne,

    No, I’m Jim Hardy. Dr Hartley is now a PI at NCI-Frederick, with a number of other brilliant colleagues who “left” when Invitrogen shut us down. He’s doing absolutely remarkable work on cell free protein expression systems and I continue to see him often. [see http://fredcobio.wordpress.com/2008/11/25/nature-methods-a-la-frederick/%5D

    I worked for Jil, who sadly hung up her lab coat for a career in Marketing. I would love to see someone reproduce or improve on this experiment, after 20 years!

  35. Marc on December 12, 2009 at 1:22 pm

    Thank you guys so very much, I have spent the last 2 years wondering about this issues. This website is great! Thanks! Cheers from Geneva!

  36. Suzanne on December 11, 2009 at 7:37 pm

    Thanks Jim! This is great information and I wasn’t familiar with the BRL papers.
    Are you the Jim Hartley from BRL who wrote the paper cited by Wikipedia?

    It is a very interesting paper but I have some comments.

    It would have been better if the incubations actually compared room temperature to 0C. These experiments only used cold temps. Even experiment 2, the centrifugations are all at 6C. It would have been good to see the difference between centrifugation at 6C vs. RT.

    This makes the Wiki entry inconsistent because it says “The best efficiency is achieved at room temperature.” and there is no reference for that statement at the end.

    Figure 2 is the experiment to look at DNA recovery after incubation at cold temps, comparing 0 minutes vs. 10 minutes at 0C. Most people precipitate DNA longer than 10 minutes at 0-4C. The centrifugation experiment in figure 3 goes out to 30 minutes at 4C but not the incubation experiment. The best comparison would have been to incubate 1 hr at 4C vs. 10 minutes to see if there is a true difference. One could argue that the samples in experiment 3 were incubated at 4C longer in the 20-30 minute spins (because the centrifuge was chilled) and this may have enhanced the recovery.

    In experiment 1, where all temps seem to recover the same amount of DNA at all temperatures, the centrifugation was only 10 minutes, which experiment 3 shows is obviously not optimal. So it is possible that the colder temps at -20C and -70C would have recovered more DNA than the 4C temperature if the centrifugation was longer. Ok- unlikely, but possible. We don’t really know what the outcome will be until we do the experiment.

    I do think this is a really good, interesting article. I am sure our readers will find it helpful.

    Thanks very much for reading and commenting!

  37. Jim H on December 11, 2009 at 3:50 pm

    Agh! I still cringe when people cling to the false belief that cold temperature is necessary. True, it does affect solubility if you’re letting gravity do the work, but it actually decreases yield if you’re using a microfuge due to the increased viscosity of the solution at lower temperatures. This Inconvenient Truth was published in 1985 by BRL, but is still such an icon of ethanol precipitation that the “Cold Ethanol Denialists” refuse to budge (even the scientists at BRL-> Life Tech wouldn’t change all of the technical manuals). See the Zeugin JA, Hartley JL reference in this wiki http://en.wikipedia.org/wiki/Ethanol_precipitation

    The main finding is that extending centrifugation time (instead of extended periods in -20C) maximizes the yield.

    • merajrizvi on January 25, 2012 at 9:47 am

      Hi Jim, Thanks for the valuable information. Does it really make a difference if the DNA settling down under the force of gravity or under the centrifugal force in a centrifuge? I usually keep the DNA at -20 degrees for a couple of hours after ethanol precipitation and it seems to me that the yield gets better although i have not compared the results quantitatively.

      Thanks

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