Ethidium Bromide (EtBr)

Ethidium bromide is a fluorescent dye used to visualize DNA and RNA in gels. Ethidium bromide is a DNA intercalator, inserting itself between the base pairs in the double helix. Ethidium bromide has UV absorbance maxima at 300 and 520 nm, and an emission maximum at 590 nm. The detection limit of DNA bound to ethidium bromide is 0.5 to 5.0 ng/band.

Ethidium bromide is routinely used to stain DNA in gels. It is also used to detect protein-DNA complexes in band shift assays and to observe single DNA molecules during gel electrophoresis. Staining of ssDNA or RNA is less sensitive, often requiring 10 times more nucleic acid for equivalent detection.

Ethidium bromide can be used in both post-electrophoresis staining and in in-gel staining methods. 

• Post-electrophoresis EtBr staining: Stain gel in a 0.5–1.0 µg/mL solution of ethidium bromide in deionized water for 15 to 60 min depending on the thickness of the gel (longer times have a risk of sample diffusion). To reduce background fluorescence, the gel can be destained in deionized water for 15 to 30 min. For RNA gels, reduce staining time and destaining is recommended.
• In-gel EtBr staining: Prior to casting the gel, add ethidium bromide to melted agarose to a final concentration of 0.5 µg/mL. This method may reduce the rate of migration of DNA fragments. However, in-gel staining reduces the quantity of ethidium bromide waste, and is a faster way to visualize results.

Learn more about electrophoresis staining