Preparation of Stock Solution of Ethidium Bromide (10 mg/ml)

Overview:

• 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 color.
• Ethidium bromide can detect as little as 1 ng DNA/band in agarose gel.
• It can also bind to RNA which results in a 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 needs to be protected from exposure to light.

Requirements

Reagents
> 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

Composition
10 mg/ ml ethidium bromide in water

Objective
Preparation of 10 ml of 10 mg/ml ethidium bromide solution in water

Precaution:
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.

Tip:
We recommend you to use a 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 the measuring cylinder. Moreover, you don’t need to contaminate reusable vessels (beakers).

Step 2: Mix until all ethidium bromide dissolves completely. This may take a long time.

Tip:
Use a tube rotator to mix all content if you are using a 15-ml screw-cap graduated polypropylene centrifuge tube.

Precaution:
Cover the tube with aluminum foil to protect ethidium Bromide from light exposure.

Step 3: Adjust the volume to 10 ml with Deionized / Milli-Q water.

Note:
The solution will appear red.

Storage:
Ethidium bromide is quite stable and can be stored at 4°C for many years if protected from light.

Application
> Detection of DNA and RNA on agarose gel.
> Purification of supercoiled DNA using cesium chloride – ethidium bromide gradient centrifugation method