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Preparation of 10 ml of 10 mg/ml (1%, 20000x) ethidium bromide solution in water

  1. Weigh out 100 mg ethidium bromide in a 15-ml Falcon tube.
  2. Add about half of the water and mix.
  3. Add water to the 10 mL mark

Wear gloves and lab coat at all times when handling ethidium bromide or ethidium bromide contaminated solutions, glassware, pipette tips, and so forth.

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 visualize 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 also bind to single-stranded DNA.

Tip:

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.

Tip:

Use tube rotator to mix all content if you 15-ml screw-cap graduated polypropylene centrifuge tube is used.

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. Ethidium bromide solution is ready for use.

Use:

This stock solution is 20000x concentrated compared to the final concentration in the gel (0.5 μg/ml). This means that 50 µL should be added to each liter of gel.

Ethidium bromide can be added to molten agarose gel before the gel is poured to a final concentration of 0.5 μg/ml. Alternatively, 1 μg/ml EtBR can be added to the loading buffer.

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.

Storage:

Ethidium bromide powder is quite stable at room temperature but need to be protected from exposure of light. Ethidium bromide water solution is quite stable and can be stored at 4°C for many years if protected from light.

References