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now: Mon Mar 8 20:51:23 2021 ... mod: Mon Jan 25 18:44:17 2021
Ethidium bromide is a common fluorescent stain used with double-stranded DNA. Ethidium intercalates between DNA bases. In the intercalated state, ethidium exposed to short-wave UV light (302nm) will fluoresce bright orange (595nm). Ethidium also has strong absorption of green light (510nm -- close enough for use with the blue 488nm emission line of the argon lasers commonly used in flow cytometry), again fluorescing orange. The intercalation of ethidium into double-stranded DNA causes the helix to extend and unwind. A covalently closed dsDNA circle treated with ethidium will become more positively supercoiled.
In agarose gels, ethidium bromide is usually added to the molten gel before the gel is poured to a final concentration of 0.5 μg/ml. The stock solution for ethidium bromide is 1% (10 mg/ml) made up in water and stored indefinitely in the dark at 4C. The 1% stock is then 20000x with respect to agarose gels. Alternatively, a typical 6 mm thick agarose gel can be poured and then run without ethidium and then soaked with gentle agitation in ethidium solution (also at 0.5 μg/ml) for 30 minutes to allow the ethidium to diffuse into the gel. Longer soak times are not useful as as background fluorescence will become problematic. If necessary the gel can be subsequently destained by gentle agitation in water for an additional 30 minutes. Do not destain longer to avoid loss of signal.
Note that DNA migrates more slowly in gels to which ethidium has been pre-added for two reasons: 1) the ethidium unwinds the helix making linear DNA molecules physically longer and 2) the positively charged ethidium will partially charge shield the DNA from the electrophoretic field. Also note that covalently closed circular DNA migrates dramatically differently when electrophoresed in the presence of ethidium, becoming strongly positively supercoiled.
Ethidium bromide can be used for cell cycle analysis by staining DNA in isolated nuclei as in the NusseNuclearPreparation
. The absorption/emission spectra of ethidium bromide is very similar to propidium iodide and in general they can be used interchangeably. Where ethidium and propidium are not
interchangeable is measuring cellular viability by looking for dye exclusion with intact cells. Ethidium can slowly cross the intact plasma membrane of cells, whereas the more highly charged propidium molecule has a more difficult time crossing intact membranes and thus makes a better indicator of membrane integrity.
⚠ Caution: Ethidium bromide is a strong mutagen. Always wear gloves when in contact with gels or solutions that have been exposed to ethidium bromide. Be very careful when making a stock solution from the dried powder to avoid spreading/inhaling fine ethidium bromide dust.