- Ethidium bromide (EtBr) is a commonly used fluorescent stain to visualize nucleic acid especially DNA in agarose gels.
- Ethidium bromide intercalates between DNA bases. Upon intercalation, its fluorescence increases several folds (25 fold increase when it binds DNA), much higher than the unbound ethidium bromide fluorescence, therefore, eliminates the need to wash gel to remove unbound ethidium bromide.
- When exposed to uv light, it appears bright pink/orange colour.
- Ethidium bromide can detect as little as 1 ng DNA/band in agarose gel.
- It can also bind to RNA which results in 21 fold increase in its fluorescence intensity.
- It has been reported to bind to single-stranded DNA.
Ethidium bromide powder is quite stable at room temperature but need to be protected from exposure of light.
- Ethidium bromide
- Deionized / Milli-Q water
- Equipment and disposables
- Measuring cylinder
- Conical flask/Beaker/15-ml screw-cap graduated polypropylene centrifuge tube
- Magnetic stirrer / Tube-Rotator
- 10 mg/ ml ethidium bromide in water
- Preparation of 10 ml of 10 mg/ml ethidium bromide solution in water
Wear gloves and lab coat at all times when handling ethidium bromide or ethidium bromide contaminated solutions, glassware, pipette tips, and so forth.
Step 1: Weigh out 100 mg ethidium bromide conical flask / beaker / 15-ml polypropylene centrifuge tube. Add 7 – 8 ml water.
- We recommend you to use 15-ml screw-cap graduated polypropylene centrifuge tube. Since these tubes have milliliter marks, you can adjust the solution volume without transferring the solution to measuring cylinder. Moreover, you don’t need to contaminate reusable vessels (beaker).
Step 2: Mix until all ethidium bromide dissolves completely. This may take long time.
- Use tube rotator to mix all content if you 15-ml screw-cap graduated polypropylene centrifuge tube is used.
- Cover the tube with aluminium foil to protect ethidium Bromide from light exposure.
Step 3: Adjust the volume to 10 ml with Deionized / Milli-Q water.
- The solution will appear red.
Ethidium bromide solution is ready for use.
- Ethidium bromide is quite stable and can be stored at 4°C for many years if protected from light.
- Detection of DNA and RNA on agarose gel.
- Purification of supercoiled DNA using cesium chloride – ethidium bromide gradient centrifugation method