5 Ways to Clean Up A DNA Sample

One of the most common tasks in molecular biology is cleaning up DNA from aqueous solutions to remove buffer salts, enzymes or other substances that could affect downstream applications. Examples include cleaning up PCR reactions, digests or other enzymatic treatments and cleaning up genomic or plasmid DNA contaminated with cellular proteins/debris. There are several ways to approach DNA clean-up, here are five of them.

1. Phenol-Chloroform Extraction

Phenol chloroform extraction (see Kirby, 1957), normally followed by ethanol precipitation, is the traditional method to remove protein from a DNA sample. In this procedure, the DNA solution is mixed with phenol and chloroform. The water-soluble DNA partitions into the aqueous phase, while the proteins are denatured by the organic solvents and stay in the organic phase. The aqueous phase containing the protein-free DNA can then be collected.

Advantages: A cheap and effective way to remove proteins from DNA solutions.
Disadvantages: Slow compared to most modern methods, there is a risk of phenol/chloroform carry-over into the final sample (which could inhibit downstream enzymatic reactions), chloroform and phenol are both hazardous chemicals.

2. Ethanol Precipitation

Ethanol precipitation is a tried and tested method for de-salting and concentrating DNA. 0.1 to 0.5 M monovalent cations (normally in the form of the acetate salt of sodium) is added to the DNA, along with ethanol to a final concentration of 70%. Ethanol changes the DNA structure so that the DNA molecules aggregate and precipitate from solution (see Eickbush and Moudrianakis, 1978). Since most salts and small organic molecules are soluble in 70% ethanol they stay in solution and the precipitated DNA can be separated from them by centrifugation.

Advantages: A cheap and effective way to de-salt and concentrate DNA.
Disadvantages: Time consuming and risk of ethanol carry-over into the final sample

3. Silica Column-based Kits

Column-based kits offer a convenient approach to DNA cleanup. The principle is that chaotrophic salts are added to the sample to denature the DNA by disrupting it’s hydrogen bonding. Under these conditions, the DNA will selectively bind to the silica resin in the column, allowing it to be separated from the rest of the sample. After washing the DNA is eluted from the column with a low salt solution which allows the re-naturing of the DNA, causing it to lose affinity for the silica. A good example of this technology is Qiagen’s Qiaquick series, which has several kits for agarose gel extraction, enzymatic reaction, nucleotide and PCR clean-up.

Advantages: Convenient, relatively fast and the user can process large number of samples using the vacuum manifold option.
Disadvantages: Fairly expensive, and in my experience low yields (as low as 25%) and chaotrophic salt carry-over are common.

Note: Zymo’s DNA clean-up and concentrator kit offers an alternative, based on the same silica resin technology, where the sample can be eluted in a very small volume to give high DNA concentrations.

4. Strataclean Resin

Stratagene’s StrataClean approach uses a slurry of hydroxylated silica, which (almost magically it seems) binds protein with a high affinity, while having a low affinity of DNA at near neutral pH. The slurry is added directly to the DNA sample, which is then mixed and centrifuged and the supernatant containing the protein-free DNA is collected. The protocol takes just a few minutes, although 2 or 3 clean-ups may be required to certain stubborn enzymes (details are available in the kit’s protocol).

Advantages: Very fast and cheap, no chaotrophic salts or organic washing solutions
Disadvantages: Only removes proteins. Removal of salts requires a traditional ethanol precipitation step.

5. Magnetic Beads

This approach uses magnetic beads that conditionally bind DNA and can be immobilized on a magnetic to separate the DNA from the rest of the sample and allow washing etc. Invitrogen’s ChargeSwitch technology is the best example of this I have seen. It uses magnetic beads that are positively charged, and will therefore bind to DNA, at low pH but at high pH they are negatively charged and release the DNA.

Advantages: Fast, no chaotrophic salts or organic washing solutions, very good yields and spectophotmetric purity in my experience
Disadvantages: The inital outlay for the magnets is reasonably high and the procedure is a bit tricky when handling multiple samples.

I think that covers it! If you have any more suggestions, or indeed any questions, please feel free to add a comment.

Originally published October 25, 2007. Updated and republished June 9, 2015.

15 Comments

  1. Gilson Azevedo on May 14, 2017 at 5:26 pm

    How much Ethanol 100% do I need to put in my samples (DNA and RNA plus 2.5 M ammonium sulfate) for the nucleic acids to precipitate in the solution? After, Do I need to wash the DNA with Ethanol 70%, right? How much?

    Sincerely,

    Gilson Azevedo

  2. cleaner on December 16, 2016 at 10:42 am

    Excellent way of explaining, and pleasant article to get data on the topic of my presentation subject matter,
    which i am going to deliver in institution of higher education.

  3. Abraham on August 3, 2016 at 9:48 pm

    I have one doubt… What happen if when ww are cleaning with magnetics beads and we let a little of ethanol?

  4. Grant MacGregor on August 2, 2016 at 3:36 am

    Another simple and efficient method is (spin) column chromatography using an appropriate matrix – e.g. Sephadex G-50 or Sephacryl S-200 equilibrated in 10/1 TE. If you don’t want to buy empty spin columns from Pierce etc, then you can make a quick one using a 0.5 ml Eppendorf that has a hole in the bottom made using a hypodermic needle (poke from inside to outside and don’t poke your finger !), placed inside a 1.5 ml Eppendorf. Stuff a small crumpled up piece of siliconized glass wool into the 0.5ml Eppendorf before adding ~ 400 ul of your appropriate chromatography slurry. Pre-spin to pack the column at a low G force (~ 500xg for 1 min), discard the buffer (I usually prepare the media in 10 / 1 TE), then add your sample to the middle of the packed matrix. Re-spin for 2 min at the same G-force and you’re done. This is an easy way to change salts, remove small pieces of DNA etc. The sample elutes in just about the same volume loaded.

  5. Meggie on April 15, 2016 at 2:10 am

    I’ve used silica based systems before, the Qiagen purification set specifically, and also experienced very low yields! I believe we have traced it down to the amount of time you allow the lysis step to proceed… we also found it to be to our advantage to use the lyse blue reagent… boosted our yields quite significantly!

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  7. Hala Mahmoud on January 14, 2016 at 11:59 pm

    If I used a cooling centrifuge in the Silica Column-based Kits is that will effect on DNA clean up or not ??

  8. Dirk Krid on August 3, 2015 at 7:46 pm

    Maybe you could add PEG extraction to the list ?
    Neat overview

    • Jerry Chen on April 7, 2016 at 1:25 am

      I agree totally!

  9. BeHi on May 28, 2013 at 12:56 pm

    Thanks for this fantastic overview!
    There is even another way of DNA clean-up called micro-dialysis. Many people clean their PCR products by osmosis before using them in downstream applications (e.g. sequencing).

    There are special membranes available for this (e.g. from Millipore, 0.025 micrometers pore size) which are placed on a petri-dish filled with low-TE (0.25x works well). On this membrane a drop of solution containing the PCR product is applied and incubated for up to 20 minutes (not much longer since the volume of the sample constantly decreases by evaporation). Subsequently the DNA can simply be transferred to a fresh tube or directly used in downstream applications.

    Advantages: Quick protocol (incubation for 10 minutes will suffice in most cases). Virtually no hands-on time.
    Disadvantages: Only salts are removed while proteins remain in the sample. DNA concentration increases due to evaporation and has to be re-determined afterwards. Don’t forget to mark the positions on the membrane: when processing more than one sample the drop will look all the same!

    • Sneha on December 30, 2015 at 4:28 am

      Can you give us a link for the micro dialysis of DNA. Can it be applied on extracted genomic DNA,apart from PCR products?

  10. James Ameh on February 13, 2010 at 2:30 pm

    Good analysis on 5 ways to clean up DNA.What about mitochondrial DNA extraction and ways in which this type of DNA can be clean up?

  11. richa on August 28, 2009 at 6:12 pm

    hi!
    could u please tell why 1:1 concentration is taken for phenol and chloroform ? why we dont use absolute alcohol for precipitation? how ethanol changes dna structure; how it actually precipitate the dna?

  12. jasti on May 13, 2009 at 6:06 am

    does phenol has any effect on DNA?? My DNA is getting sheared..i need longer fragments for my exp.. can you help me out?

  13. Susana on January 30, 2009 at 6:42 am

    Why is it that in some procedures one has to add 100% ethanol first and then the 70% ethanol? What would be the purpose of the 100% ethanol?

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