Ethidium bromide is a highly sensitive fluorescent stain used to observe DNA in agarose and polyacrylamide gels. Ethidium bromide is excited by a standard 302nm ultraviolet light transmission instrument, and emits an orange-red signal, which can be photographed by a polaroid negative or gel imaging processing system with a CCD imaging head.

The most common way to observe DNA in agarose gel is to dye it with fluorescent dye ethidium bromide, which contains a tricyclic planar group and can be embedded between DNA stacking bases. Its binding to DNA has almost no base sequence specificity. In saturated solution with high ionic strength, an ethidium bromide molecule is inserted about every 2.5 bases. When the dye molecule is inserted, its plane group is perpendicular to the helix axis, and it interacts with the upper and lower bases through van der Waals force. The fixed position of the group and its close proximity to the base lead to the fluorescence of the dye bound to DNA, and its fluorescence yield is higher than that of the dye in free solution. DNA absorbs 254 nm ultraviolet radiation and transmits it to the dye, while the combined dye itself absorbs 302 nm and 366 nm optical radiation. In both cases, the absorbed energy is re-emitted at 590nm in the red-orange region of the visible spectrum. Because the fluorescence yield of ethidium bromide -DNA complex is 20-30 times higher than that of dyes without DNA binding, as few as 10ng of DNA bands can be detected when the gel contains free ethidium bromide (0.5ug/ml).

Ethidium bromide can be used to detect single-stranded or double-stranded nucleic acids (DNA or RNA). However, the affinity of dyes for single-stranded nucleic acids is relatively small, so their fluorescence yield is relatively low. In fact, most of the fluorescence of single-stranded DNA or RNA is produced by the binding of dyes to molecules to form short intramolecular helices.

Although the electrophoretic mobility of linear DNA is reduced by about 15% in the presence of this dye, when the exact size of DNA fragment needs to be known (such as the identification of DNA restriction enzyme digestion map), the gel should be stained with EB without EB electrophoresis. Decolorization is generally not required after dyeing. However, when detecting a small amount of DNA fragments (less than 10ng), it is usually necessary to decolorize the dyed gel.