Ethidium bromide (also abbreviated EtBr) is an nucleic acid intercalating agent often used as a nucleic acid stain in molecular biology research for protocols including agarose gel electrophoresis.
When exposed to UV (ultraviolet light), ethidium bromide fluoresces with a red-orange color, intensifying almost 20-fold after binding to DNA. Ethidium bromide is also a very strong mutagen, and may possibly be a carcinogen or teratogen.
Applications of Ethidium Bromide in the Laboratory
In the case of DNA this is usually double-stranded DNA from PCRs, restriction digests, etc. Single-stranded RNA can also be detected, since it usually folds back onto itself and thus provides local base pairing for the dye to intercalate.
Detection typically involves a gel containing nucleic acids placed on or under a UV lamp. Since ultraviolet light is harmful to eyes and skin, a camera is used for recording ethidium bromide fluorescence. In other cases, a protective screen is put between the observer and the UV source. In the cytogenetic laboratory the intercalating properties have long been utilized to minimize chromosomal condensation when a culture exposed to mitotic arresting agents during harvest. The resulting slide preparations permit a higher degree of resolution and thus confidence, in determining structural integrity of chromosomes upon microscopic analysis.
Alternatives to Ethidium Bromide
There are alternatives to ethidium bromide in the lab. Several SYBR or or SYBR Safe stains are used by some as a safer alternative. SYBR dyes have been found to be less carcinogenic than EtBr and to give cleaner, higher powered staining. However, they are suspended in DMSO which can rapidly pass through skin. However, many researchers still prefer ethidium bromide for difficult detections. Note that Sigma-Aldrich claims a similar detection limit for the SYBR stain.
Safety Precautions when using Ethidium Bromide
Spills or personal exposure
As with any chemical, if spilled on skin or eyes, rinse for 15 minutes using a safety shower or eyewash. If inhaled or swallowed, seek medical attention immediately.
For spills, use a spill pillow or absorbent to soak up aqueous ethidium bromide. Carefully clean up solid ethidium bromide to avoid creating dusts. Place in a sealable container and dispose in a medical waste box.
Clean the spill carefully with ethanol, and then the solution must be treated with activated charcoal.
All solutions containing ethidium bromide must be decontaminated before being discarded.
To clean with Activated Charcoal:
1. Add 100 mg activated charcoal/ ml dye solution.
2. Let stand at least 1 hour with occasional stirring.
3. Filter through Whatman No.1 filter paper.
4. Pour the clear filtrate into the sink.
5. Place the filter paper containing the dye/charcoal mixture into the biohazard waste.
Waste disposal of Ethidium Bromide
Ethidium bromide should be handled and disposed of as HAZARDOUS waste. This applies to gloves, test tubes, paper towels, etc., that are grossly contaminated with ethidium bromide as well. All ethidium bromide waste should be considered state regulated hazardous waste.
Safe handling of Ethidium Bromide
Preparation of stock solutions and any operations capable of generating ethidium bromide dust or aerosols should be conducted in a fume hood to prevent inhalation. Gloves should be worn at all times. Latex gloves offer little protection from ethidium bromide – gloves made of nitrile rubber are much more effective. When working with high concentrations (stocks or powder) or for a prolonged period of time, double gloving can further reduce the risk of exposure, especially if the outer glove is replaced whenever significantly contaminated.
Ethidium bromide (10 mg/mL) Recipe
Dissolve 0.2 grams of ethidium bromide in 20 mL of H2O
Mix and store at 4°C wrapped in aluminum foil (dark).
NOTE: Use Caution - Ethidium bromide is a mutagen, and must be handled very carefully.
