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Decontamination of Ethidium Bromide

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ORS - Chemical Safety


Decontamination of Ethidium Bromide

Decontamination of Concentrated Solutions (over 0.5 mg/ml) 

Note: Treatment of ethidium bromide solutions with bleach is not permitted. 

Method 1 ( LUNN AND SANSONE 1987) 

1. Add sufficient water to reduce the concentration of ethidium bromide to 0.5mg/ml or less. Work in a chemical fume hood. 

2. For each 100 ml of ethidium bromide solution, add 20 ml of fresh 5% hypophosphorous acid and 12 ml of fresh 0.5 molar sodium nitrite. Mix carefully. 

Important: Check that the pH of the solution is less than 3.0 

Hypophosphorous acid is usually supplied as a 50% solution, which is corrosive and should be handled with care. It should be freshly diluted before use: 10 ml of hypophosphorous acid into 90 ml of water, stirred briefly. Use a chemical fume hood to prepare this solution. 

Sodium nitrate solution, 0.5 molar, should be freshly prepared by dissolving 3.45 grams of sodium nitrite in water to a final volume of 100 ml. Use a chemical fume hood to prepare this solution. 

3. After incubaton for 24 hours at room temperature, bring the pH to between 5 and 9 with sodium bicarbonate. Discard the solution to the drain. 

Method 2 (Quillardet and Hofnung 1988) 

1. Add sufficient water to reduce the concentration of ethidium bromide to 0.5 mg/ml or less. work in a chemical fume hood. 

2. To each 100 ml of ethidium bromide solution, add 100 ml of 0.5 molar potassium permanganate solution followed by 100 ml of 2.5 normal hydrochloric acid. Mix carefully and allow the solution to stand at room temperature for several hours. 

Dilute 7.9 grams of potassium permanganate in water and bring the volume to 100 ml to make a 0.5 molar solution. 

Add 20.7 ml of concentrated hydrochloric acid to water and bring the volume to 100 ml to make a 2.5 normal solution. 

3. Prepare a sodium hydroxide solution, about 2.5 normal by dissolving 10 grams of sodium hydroxide in water and bringing the volume to 100 ml. Slowly add the 2.5 nornal solution to bring the pH to between 5 and 9. Discard the liquid down the drain. 

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Decontamination of Dilute Solutions (Up to 100 ug/ml) 

Method 1 (Lunn and Sansone 1987) 

1. Add 2.9 g of Amberlite XAD-16 for each 100 ml of solution up to 100 ug/ml ethidium bromide. Amberlite XAD-16, a nonionic, polymeric absorbant, is available from Rohm and Haas and sold by Sigma Chemical Company. 

2. Store the soltion for 12 hours at room temperate, shaking it intermittently. 

3. Filter the solution through a Whatman No. 1 filter. Discard the filtrate to the drain. 

4. Seal the filter and Amberlite in a plastic bag and dispose of the bag through the Office for Research Safety. 

Method 2 (Bensaude 1988) 

1. Add 300mg of powdered activated charcoal for each 100 ml of up to 10 ug/ml solution of ethidium bromide. 

2. Store the solution for 1 hour at room temperature, shaking it intermittenly. 

3. Filter the solution through a Whatman No. 1 filter. Discard the filtrate to the drain. 

4. Seal the filter and activated charcoal in a plastic bag, and dispose of the bag through the Office for Research Safety. 

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Decontamination of Ethidium Bromide Spills 

Method 1 (Lunn and Sansone 1989) 

1. De-energize all electrical equipment before decontamination. Wear appropriate protective equipment including gloves, lab coat, and safety goggles. 

2. Absorb all free liquid from the spill with paper towels. 

Caution: The towels may contain fairly concentrated ethidium bromide solutions. 

3. Scrub the contaminated surface six separate timess, each time using a fresh, wet paper towel. Place all towels into a container. 

4. In a chemical fume hood, dissolve 4.2 grams of sodium nitrite in 300 ml water and then add 20 ml hypophosphorous acid (50%). Stir briefly to ensure mixing. The pH of the mixture should be around 1.8. 

5. In the hood, pour the freshly made decontamination mixture described in No. 3 over the towels and soak them for at least 1 hour. 

6. Test the used decontamination solution by fluorescence spectrophotometry. Squeezings from towels from some of the later washes should also be tested 

7. Dry the equipment or surface and place into service. Bring the pH of the towels/solution to between 5 and 9 with sodium bicarbonate. Discard the free liquid to the drain. Place the wet towels into a sealed plastic bag and discard in the normal trash. 

Method 2 (Bensaude 1988) 

1. Wipe up excess liquid with paper towels. Wet surface with ethanol and sprinkle activated charcoal on the surface. 

2. Wipe up the charcoal/ethanol mixture with paper towels. Seal the wastes in a plastic bag and dispose of the bag through the Office for Research Safety. 

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References 

Bensaude, O. Ethidium Bromide and Safety- Readers Suggest Alternative Solutions. Trends Genet. 4:89; 1988. 

Lunn, G.; Sansone, E.B. Ethidium Bromide: Destruction and Decontamination of Solutions. Anal. Biochem. 162:453; 1987. 

Lunn, G.; Sansone, E.B. Decontamination of Ethidium Bromide Spills. Appl. Ind. Hyg. 4: 234-237; 1989. 

Quillardet, P.; Hofnung, M. Ethidium Bromide and Safety- ReadersSuggest Alternative Solutions. Trends Genet. 4:89.


 

 

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