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5 Ways to Clean Up A DNA Sample

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One of the most common tasks in molecular biology is DNA clean up. We clean up DNA from aqueous solutions to remove buffer salts, enzymes or other substances that could affect downstream applications. Examples include PCR reaction clean up, clean up after restriction digests and clean up of genomic or plasmid DNA contaminated with cellular proteins/debris.

Conveniently, there are several ways to clean up DNA. 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 denature in the presence of organic solvents, thus staying in the organic phase. The aqueous phase containing the protein-free DNA is then ready for collection.

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 popular method for desalting and concentrating DNA. Monovalent cations (0.1 to 0.5 M, normally in the form of the acetate salt of sodium) are 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 out of solution (see Eickbush and Moudrianakis, 1978). Most salts and small organic molecules are soluble in 70% ethanol, leaving the precipitated DNA ready for separation by centrifugation.

Advantages: A cheap and effective way to desalt and concentrate DNA.
Disadvantages: Time consuming. You risk carrying ethanol over into the final sample.

3. Silica Column-Based Kits

Column-based kits offer a convenient approach to DNA cleanup. The principle is that chaotropic salts are added to the sample to denature the DNA by disrupting its hydrogen bonds. Under these conditions, the DNA will selectively bind to the silica resin in the column, facilitating its separation from the rest of the sample. After washing, the DNA is eluted from the column with a low salt solution that allows renaturing, causing the DNA to lose affinity for the silica. Most commercial suppliers offer kits based on this technology, with a range of kits available for DNA cleanup after agarose gel extraction, enzymatic reactions and PCR, to name a few.

Advantages: Convenient, relatively fast and the user can process a large number of samples using the vacuum manifold option.
Disadvantages: May be costly. Some kits give low yields (as low as 25%). Chaotropic salt carryover may be an issue.

Note: Some suppliers are now offering improved silica column kits, where sample elution takes place in a very small volume to yield high DNA concentrations.

4.  Anion Exchange

Anion exchange DNA purification methods usually use positively charged DEAE functionalized resins to bind the negatively charged DNA phosphate backbone. Using specific salt and pH conditions, DNA in the sample binds the resin, and stringent washing steps remove contaminants (e.g. protein, cellular debris). The remaining DNA can then be selectively eluted from the resin.

Advantages: high purity DNA for downstream applications e.g. transfection and DNA sequencing.
Disadvantages: resins can be expensive.

5. Magnetic Beads

This approach uses magnetic beads that conditionally bind DNA in a pH-dependent manner, allowing you to separate the DNA from the rest of the sample by simply controlling pH. There are a few magnetic bead kits on the market. The magnetic beads are positively charged and bind DNA at low pH, but at high pH they become negatively charged, thus releasing the DNA.

Advantages: Fast, no chaotrophic salts or organic washing solutions required. Ideal for automation of high throughput processing, since there is no need for centrifugation and other time consuming processing steps. Note there are also magnetic bead kits that selectively bind DNA depending on salt concentration.
Disadvantages: The initial cost in purchasing the magnets is reasonably high. This procedure can be tricky when handling multiple samples, but this is not an issue if you have access to an automated workflow.

Originally published October 25, 2007. Updated and republished June, 2019.

Image Credit: Michael Dolan

16 Comments

  1. 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.

  2. 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!

  3. the brit method on February 4, 2016 at 5:28 am

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  4. 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 ??

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

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

  6. 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?

  7. 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?

  8. 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?

  9. 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?

  10. 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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