Ethidium Bromide

What is ethidium bromide?

Ethidium bromide (EtBr) is a fluorescent dye widely used in molecular biology research. Early usage was as a veterinary trypanocide.¹⁴ It is a mutagenic compound that intercalates double-stranded DNA and RNA.^1,10 The fluorescence of EtBr increases 21-fold upon binding to double-stranded RNA, 25-fold on binding double-stranded DNA (although histones block binding of EtBr to DNA). Ethidium bromide has been used in multiple fluorimetric assays for nucleic acids.^15,16,17 It has been shown to bind to single-stranded DNA (although not as strongly)² and triple-stranded DNA.¹⁸ Because of the binding to DNA, EtBr is a powerful inhibitor of DNA polymerase.⁴

• Molecular Biology-grade Powder (Product No. E7637) is suitable for use in gel electrophoresis and DNA isolation procedures.
• Aqueous Solution (10 mg/mL) (Product No. E1510) is suitable for use in gel electrophoresis and DNA isolation procedures.
• Molecular Biology-grade Aqueous Solution (500 mg/mL) (Product No. E1385) is suitable for use in gel electrophoresis.

Synonyms: 3,8-Diamino-5-ethyl-6-phenylphenanthridinium bromide; 2,7-Diamino-10-ethyl-9-phenylphenanthridinium bromide; Homidium Bromide; Dromilac; EtBr

Ethidium bromide product details

Appearance: Red to purple powder
Molecular formula: C₂₁H₂₀N₃Br
Molecular mass: 394.3
Melting point: 255-266 °C with decomposition^1,2

Spectral data:
UV/Vis Absorbance:
Maxima at 210 nm, E^mM = 0.20-0.5; 285 nm, E^mM = 5.0-10.0; 316 nm, E^mM = 50.0; 343 nm, E^mM = 40.0 (water)³
Published absorption spectrum (methanol) with λ_max at 296 nm and
525 nm¹
λ_max = 475 nm, E^mM = 5.76 (0.66 M glycine buffer)⁴

Solvent effects on absorbance:
λmax = 480 nm (water); 515 (glycerol); 520 nm (methanol): 520 nm (acetone); 532 nm (ethanol); 535 (DMSO) and 540 nm (pyridine).5 The absorbance maximum relative to water is red-shifted to longer wavelengths upon binding to nucleic acids.6,7

Fluorescence
Excitation/emission wavelengths reported for the EtBr-nucleic acid complex:
Ex at 526 nm, Em at 605 nm (aqueous)8
Ex at 360 nm, Em at 590 nm (in PBS)9
Ex at 525 nm, Em at 600 nm (10 mM TBE, pH 8.0)10
Ex (absorption) at 510 nm, Em at 590 nm.11

Ex (absorption) at 482 nm (blue-green), Em at 616 nm (red-orange).12 The fluorescence yield of EtBr increases as solvent polarity decreases.13

Storage: The solid powder has shown minimal change after two years stored at room temperature, protected from light.

The recommended ultimate disposal method is by incineration as discussed in the SDS.23 Processing solutions through Rohm and Haas Amberlite XAD-16 resin was shown to be effective to the limit of detection, with none of the completely decontaminated solution found to be mutagenic.24,25 We offer XAD-16 as a bulk product, as well as several products that adsorb the dye for easier disposal. See Rezorian A161 cartridges (Product No. 57611 or 57610) and the Extractor™ for EtBr decontamination (Product No. Z361569). A 5 mL Rezorian A161 cartridge can be used to treat more than 16 liters of solution (0.5 mg/liter of EtBr) before dye breakthrough. The luer lock cartridge can be used with 4 mm I.D. tubing, syringes or low-pressure chromatographic systems. The Extractor™ for EtBr decontamination can process up to 10 liters of solution (0.5 mg/liter of EtBr). Other methods of treatment for disposal of aqueous EtBr solutions have been suggested.26,27,28 We have not verified and do not endorse these procedures.

Related Products

Methylene blue (Product No. M4159) was reported as an alternative stain.19 Thiazole orange homodimer (monomer = Product No. 390062) was noted as nonmutagenic, but useful for detecting DNA in agarose gels.20

For use in cell studies, dihydroethidium (Product No. D7008), also known as hydroethidine, is a possible alternative. It is the reduced form of the dye and is converted (dehydrogenated) inside the cell to the ethidium cation, which then intercalates into DNA.21,22

Preparing ethidium bromide

At room temperature, EtBr dissolves in water at 10 mg/mL to give a red solution.2 It should be soluble up to 20 mg/mL in water or up to 2 mg/mL in ethanol.1 It is soluble at 1 part in 750 parts chloroform.3

Stock solutions of EtBr in water or PBS are stable for at least two years at room temperature if protected from light.11

Electrophoresis staining with ethidium bromide

Use in Electrophoresis Staining6

1. The dye is usually incorporated into the gel and the electrophoresis buffer at 0.5 mg/mL. Note: Electrophoresis mobility of linear double-stranded DNA is reduced by approximately 15 % in the presence of the dye.
2. To stain after gel has been run, immerse gel in electrophoresis buffer or water containing EtBr (0.5 mg/mL) for 30-45 minutes at room temperature.
3. Destaining is optional. Detection of small amounts (<10 ng) of DNA is made easier if the background fluorescence caused by unbound EtBr is reduced by soaking the stained gel in water or 1 mM MgSO4 for 20 minutes at room temperature.

References

1. Green, F. 1990 . Sigma-Aldrich Handbook of Stains, Dyes and Indicators Aldrich Chemical Co . p. 318..

2. Internal quality control - molecular biology laboratories..

3. 1996 . "Merck Index . 12th ed., #4767.

4. Elliott W. 1963. The effects of antimicrobial agents on deoxyribonucleic acid polymerase. 86(3):562-567. https://doi.org/10.1042/bj0860562

5. Olmsted J, Kearns DR. 1977. Mechanism of ethidium bromide fluorescence enhancement on binding to nucleic acids. Biochemistry. 16(16):3647-3654. https://doi.org/10.1021/bi00635a022

6. Sambrook, Jea. 1989 . Molecular Cloning: A Laboratory Manual . 2nd ed, p 6.15, 6.44 . Cold Spring Harbor Laboratory.

7. Le Pecq J. Use of Ethidium Bromide for Separation and Determination of Nucleic Acids of Various Conformational Forms and Measurement of Their Associated Enzymes.41-86. https://doi.org/10.1002/9780470110393.ch2

8. "Bechtol, Kea. December 1994 . American Biotechnology Lab . p. 8.

9. Boer G. 1975. A simplified microassay of DNA and RNA using ethidium bromide. Analytical Biochemistry. 65(1-2):225-231. https://doi.org/10.1016/0003-2697(75)90507-2

10. Severini A, Richard Morgan A. 1991. An assay for proteinases and their inhibitors based on DNA/ethidium bromide fluorescence. Analytical Biochemistry. 193(1):83-89. https://doi.org/10.1016/0003-2697(91)90046-v

11. Crary, B, Boyrsenko, M. 1986 . Biotechniques . 4, 98-101.

12. Givan, A. Flow Cytometry: First Principles . p. 104 and p. 64,. Wiley-Liss, NY :

13. 1974 . Methods in Enzymology . 32, 239.

14. Watkins TI. 1952. 585. Trypanocides of the phenanthridine series. Part I. The effect of changing the quaternary grouping in dimidium bromide. J. Chem. Soc..3059. https://doi.org/10.1039/jr9520003059

15. Moore SP, Sutherland BM. 1985. A densitometric nondestructive microassay for DNA quantitation. Analytical Biochemistry. 144(1):15-19. https://doi.org/10.1016/0003-2697(85)90077-6

16. El-Hamalawi AA, Stuart Thompson J, Barker GR. 1975. The fluorometric determination of nucleic acids in pea seeds by use of ethidium bromide complexes. Analytical Biochemistry. 67(2):384-391. https://doi.org/10.1016/0003-2697(75)90309-7

17. Karsten U, Wollenberger A. 1977. Improvements in the ethidium bromide method for direct fluorometric estimation of DNA and RNA in cell and tissue homogenates. Analytical Biochemistry. 77(2):464-470. https://doi.org/10.1016/0003-2697(77)90259-7

18. Scaria PV, Shafer RH. 1991. Binding of ethidium bromide to a DNA triple helix. Evidence for intercalation.. Journal of Biological Chemistry. 266(9):5417-5423. https://doi.org/10.1016/s0021-9258(19)67611-8

19. 1989 . Technique: A Journal of Methods in Cell and Molec. Biol . 1, 183-187 . Philadelphia, PA : Saunders Scientific Publications.

20. Wilke, Wea. 1994 . Mod. Pathol . 7(3):385-387 .

21. Quantitative Fluorescent Microassay for Identification of Antiproliferative Compounds2. https://doi.org/10.1093/jnci/77.6.1235

22. Budd SL, Castilho RF, Nicholls DG. 1997. Mitochondrial membrane potential and hydroethidine-monitored superoxide generation in cultured cerebellar granule cells. 415(1):21-24. https://doi.org/10.1016/s0014-5793(97)01088-0

23. Corresponding Safety Data Sheet (SDS)..

24. Joshua H. 1986 . Quantitative adsorption of ethidium bromide from aqueous solution by macroreticular resins . BioTechniques . 4 207–208.

25. Lunn G, Sansone EB. 1991. Decontamination of Aqueous Solutions of Biological Stains. Biotechnic & Histochemistry. 66(6):307-315. https://doi.org/10.3109/10520299109109992

26. Lunn G, Sansone EB. 1987. Ethidium bromide: Destruction and decontamination of solutions. Analytical Biochemistry. 162(2):453-458. https://doi.org/10.1016/0003-2697(87)90419-2

27. 1992 . Communications from M.A. Armour . University of Alberta, Edmonton, Canada – prior to presentation at First Asian Symposium on Academic Activity for Water Treatment Tokyo";

28. ZOCHER R, BILLICH A, KELLER U, MESSNER P. 1988. Destruction of Ethidium Bromide in Solution by Ozonolysis. Biological Chemistry Hoppe-Seyler. 369(2):1191-1194. https://doi.org/10.1515/bchm3.1988.369.2.1191