Ethidium Bromide: The Alternatives

alternative.jpgLast week, in my article about the perils of exposing DNA to UV light during cloning procedures, I mentioned a couple of stains that offer an alternative to ethidium bromide for DNA visualisation.

I this article I compare all of the available DNA stains (I know of) that can be used in electrophoresis to clarify the options available to you.

Ethidium Bromide

The classic DNA stain. Ethidium bromide is a flat molecule that fits between adjacent base pairs (intercalates) in the DNA double helix. It has UV absorbance maxima at 300 and 360nm, and can also absorb energy from nucleotides excited at 260nm. The absorbed energy is emitted as orange/yellow light at 590nm. The fluoresence 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 1-5ng
Toxicity: Toxin, mutagen, tetratogen and carcinogen according to a variety of tests but effects on higher organisms have not been proven (see here)
Price: 0.00075 GBP/100mL gel (based on a cost of 15 GBP/g EtBr)
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 bind ionically to DNA and RNA. Since it's interaction with DNA/RNA is weak, methylene blue is not a very sensitive stain, but 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: 40-100ng bands are reported to be detectable after de-staining. In my own experience, only bands of 500ng and over are reliably detectable.
Toxicity: Non-mutagenic. Somewhat toxic if ingested.
Price: 0.000015 GBP/100mL of staining solution (based on a cost of 0.6 GBP/g)
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 apparently less mutagenic. It's 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: 100-200ng bands are reported to be detectable, but according to DK's comment here on Bitesize Bio, 2ug+ may be required for clearly visible bands.
Toxicity: Mutagen (less so than ethidium bromide)
Price: 0.03 GBP/100mL gel (based on a cost of 0.28 GBP/g)
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 being less harmful than ethidium bromide, but this is debatable. It's major advantage is that it is as sensitive as ethidium bromide but does not require UV light for visualisation.
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. You can read the white paper on SYBR safe toxicity here.
Price: 0.96 GBP/100mL gel (Based on a cost of 38.70 GBP / 400ul of 10,000X SYBR safe)
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- less mutagenic than ethidium bromide, but more stable in storage than SYBR safe. Like ethidium bromide, Gel Red is visualised 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 300nm, emission at 595nm – so conventional UV transilluminators are sufficient.
Sensitivity: Bands of 0.25ng can be detected, according to my calculations from the data available in the product brochure.
Toxicity: Less mutagenic than ethidium bromide. Read the report here.
Price: 1.9GBP/100mL gel (Based on a cost of ?19/100ul of 10,000x gel red)
Reference: Biotium's Gel Red product page

Are you using a stain that I have not mentioned? Leave a comment.

Photo: Nick Dimmock

About the author: Nick Oswald
I started Bitesize Bio on a Macbook on my kitchen table in 2007 while in my 7th year of working as a molecular biologist in biotech. My aim was to share the know-how that I had acquired from the school of hard-knocks in the lab, so that others could learn from my mistakes and small victories. Nowadays my mission is to facilitate the gathering of hardcore know-how from whole spectrum of bioscientists and share it here on Bitesize Bio to create a super-mentor that any bioscientist can turn to for much-needed guidance.

See more from Nick Oswald Visit their website Twitter

7 thoughts on “Ethidium Bromide: The Alternatives”

  1. Erica says:

    Hi Nick,

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

    Thanks,
    Erica

  2. Sandy says:

    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.

  3. David J. Heinrich says:

    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.

  4. Avatar of Nick Oswald Nick Oswald says:

    David – Thanks for this – very well put and I agree entirely. I quoted rosie redfield's article in this article on bitesize bio: http://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.

  5. Jeremy says:

    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.

  6. Lindsey Kayman says:

    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. Pingback: ??????

Speak Your Mind

Nucleic Acid Purification and Analysis