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Ethidium Bromide: The Alternatives

Ethidium Bromide alternatives

How can you avoid the perils of exposing DNA to UV light during cloning procedure? Use an alternative DNA stain! Ethidium bromide is not your only option. In this article, we will compare the available DNA stains that can be used in electrophoresis to clarify the options available to you.

Ethidium Bromide

The classic DNA stain. Ethidium bromide (EtBr) is a flat molecule that fits between adjacent base pairs (intercalates) in the DNA double helix. It has UV absorbance maxima at 300 nm and 360 nm and can also absorb energy from nucleotides excited at 260 nm. The absorbed energy is emitted as orange/yellow light at 590 nm. The fluorescence of EtBr is significantly higher when intercalated than it is in aqueous solution.
Protocol: Can be used in the gel at or as a post-stain at a concentration of 0.5 mg/L
Detection: UV light
Sensitivity: Can detect bands of 1–5 ng
Toxicity: Toxin, mutagen, tetratogen and carcinogen according to a variety of tests but effects on higher organisms have not been proven (see here)
References: Karsten, U. and Wollenberger, A. Anal. Biochem. 77, 464-470, (1977)

Methylene Blue

Methylene blue is member of the thiazin family of dyes that ionically bind to DNA and RNA. Because its interaction with DNA/RNA is weak, methylene blue is not a very sensitive stain. However, it has the advantage that is detectable in the visible range. Destaining in water may be required for maximum sensitivity.

Protocol: Post strain only, in 0.025% (w/v) methylene blue in water.
Detection: Visible light.
Sensitivity: 40–100 ng bands are reported to be detectable after de-staining.
Toxicity: Non-mutagenic. Somewhat toxic if ingested.
References: Yung-Sharp, D. and Kumar, R. (1989) Technique 1 (3) 183-187.

Crystal Violet

Crystal violet intercalates into DNA in a similar manner to ethidium bromide but is less mutagenic. Its major advantage is that it is detectable in the visible range—so, no need for UV exposure.
Protocol: Use in gels at a concentration of around 1.2 mg/mL
Detection: Visible light
Sensitivity: 100–200 ng bands are reported to be detectable
Toxicity: Mutagen (less so than ethidium bromide)
Reference: Rand, K.N. Elsevier Trends Journals Technical Tips, Online, T40022, 1996.

SYBR Safe

SYBR safe is a commercial DNA stain manufactured by Invitrogen. It is marketed as less harmful than ethidium bromide, but this is debatable. Its major advantage is that it is as sensitive as ethidium bromide but does not require UV light for visualization.
Protocol: SYBR safe is used as an in-gel stain only. It is supplied in ready-made buffers so the working concentration is unknown.
Detection: For visualising fragments required for downstream applications, the best (although more expensive) option is to use a blue light box as the wavelengths used do not cause DNA damage. UV-transilluminators can also be used, although specific filters may be required.
Sensitivity: As sensitive as ethidium bromide – bands of 1-5ng should be detectable.
Toxicity: Documented as less mutagenic that ethidium bromide, but it’s acute toxicity is higher
Reference: Invitrogen’s SYBR safe product page

Gel Red

Gel Red is a commercial DNA stain manufactured by Biotium. It is marketed as being the most safe, sensitive and robust nucleic acid gel stain. Also, it is less mutagenic than ethidium bromide and more stable in storage than SYBR safe. Like ethidium bromide, Gel Red is visualized using UV light.
Protocol: Gel red can be used as post stain or in-gel stain. It is supplied in ready-made buffers so the working concentration is unknown.
Detection: UV: excitation at 300 nm, emission at 595 nm—so conventional UV transilluminators are sufficient.
Sensitivity:Bands of 0.25 ng can be detected.
Toxicity: Less mutagenic than ethidium bromide.
Reference: Biotium’s Gel Red product page

Image Credit: Diariocritico de Venezuela

13 Comments

  1. James on March 6, 2018 at 6:45 pm

    Hi,

    Biotium put together an interesting comparison of many popular ethidium bromide alternatives, looking at cell permeability, safety, and sensitivity. You can find it at the following link:
    https://biotium.com/wp-content/uploads/2017/02/Gel-Stains-Comparison.pdf

  2. Daniel on October 2, 2017 at 4:17 pm

    Methyl green is also an alternative I think it should be considered. It is much safer under the Ames’ test, and its price is very low. Worth a try.
    https://link.springer.com/article/10.1007/s00418-014-1215-0
    Best,
    Daniel

  3. joanne on May 16, 2017 at 11:49 am

    I’m using Midori Green. Any word on that?

  4. El on December 8, 2016 at 11:34 am

    What about peqGreen? Do you have any experience using it?

    • Dirk on December 19, 2016 at 12:13 pm

      Hello EL,
      peqGreen works very well. I´ve been using it since Feb 2016. Usually I use 1µl per ml of agarose for a 30ml gel. The gel shouldn´t be too thick so I reduced my gel vol from 50ml to 30ml.
      Best wishes
      Dirk

      • sofia on January 19, 2017 at 2:49 pm

        Hello Dirk,
        Do you know the coshh form for the peqgreen?
        Thank you in advance,
        Sofia

  5. dau.cc on January 4, 2016 at 10:50 pm

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    while I was browsing on Yahoo for something else, Anyways I am here now and would just like to
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  6. Lindsey Kayman on February 23, 2009 at 4:23 pm

    Hello,
    Are you familiar with the Ethidium Bromide alternative EZ Vision
    http://www.bioexpress.com/index.html?wscdet_show=000000000100112000112025

    I was wondering what your experience has been with it.
    Thank you.

  7. Jeremy on February 3, 2009 at 7:52 pm

    Nile Blue A (sigma):

    Make a 10,000x (10 mg/ml) stock in water. Put it in both running buffer and gel (10 ul stock per 100 ml gel). higher concentrations will change the migration.

    visualize on white light box. Can detect ~ 50 ng bands. If you post-stain for 1h with 10x higher concentration, you’ll detect ~ 5ng bands.

    the absorbance max of the dye is ~ 600 nm, so if you can filter the illumination to this region, you’ll increase sensitivity. For preparative gels, these detection levels are usually fine.

  8. Nick on March 11, 2008 at 9:44 am

    David – Thanks for this – very well put and I agree entirely. I quoted rosie redfield’s article in this article on bitesize bio: https://bitesizebio.com/2007/09/26/ethidium-bromide-a-reality-check/

    My only concern with EtBr is the need for UV light for visualisation, which is not so good for cloning so I use methylene blue staining for DNA I wish to use for downstream applications.

  9. David J. Heinrich on March 11, 2008 at 12:37 am

    I think that the fears of Ethidium Bromide are over-done.

    Take a look at this article on the heresy of doubting EtBr\’s toxicity:

    http://rrresearch.blogspot.com/2006/10/heresy-about-ethidium-bromide.html

    \”there\’s no direct evidence that exposure to EthBr causes mutations, tumors or birth defects in any animal, and its routine use at high doses in cattle suggests that it doesn\’t.\”

    Also, Sybr Safe is by many measures much more toxic to living cells than EtBr. Go figure.

    Now, that doesn\’t mean people shouldn\’t use Sybr Safe over EtBr. As the article I referenced notes, Sybr Safe Gold is much more sensitive, thus may be cheaper ultimately (even though the reagent itself is more expensive), because it allows the use of smaller quantities of expensive DNA standard ladders (and also less PCR reagents, restriction enzyme reagents, etc).

    Just because something might be dangerous, doesn\’t mean it should be feared with irrationality. I bet more people are severely injured by Bunsen burner flames than by any staining dye. Yet, few people have this irrational fear of Bunsen burners. The appropriate way to deal with potentially toxic chemicals is to treat them carefully, wear appropriate gloves (e.g., nitrile for EtBr, as EtBr is permeable to latex), and dispose of waste properly. The way to deal with that *is not* to spend 126,666 THOUSAND times more money (per 100ml of staining solution) to get Gel Red.

  10. Sandy on March 9, 2008 at 9:17 pm

    I worked in a human genetics lab for a while. We switched from ethidium to gel red while I was there, and I loved it. We got much better images; plus, I would occasionally set up gels without gloves just because I could.

  11. Erica on March 3, 2008 at 6:50 pm

    Hi Nick,

    FYI–We use GelStar nucleic acid stain from Cambrex. It is visualized using UV light. Works well–

    Thanks,
    Erica

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