DNA extraction in dye processing technology

EtBr is a mutagenic substance, if touched or improperly cleaned, there is a risk of EtBr contamination.

This is a substance used in molecular biology laboratories, mainly used to stain (label) DNA/RNA during electrophoresis. There are currently the following ways to use:

Direct addition of EtBr to the electrophoresis buffer.

EtBr was added to the sample loading buffer.

+ EtBr is added to the gel, when it is still not solid, then shaken well, this way the DNA (load sample) is still stained well.

Some electrophoresis processes stain the DNA after electrophoresis.

EtBr removal methods:

Requirements for EtBr waste:

2.1. Gel electrophoresis containing EtBr:

All gels and solids containing EtBr should be collected and removed from the work environment as hazardous waste.

Must be packaged separately, clearly marked as waste containing EtBr.

2.2. Ethidium bromide solution:

Buffers containing more than 0.5 mg/ml EtBr should be filtered, reduced or burned, or stored as hazardous chemical waste.

When the solution is less than 0.5mg/ml can be disposed of through the drain.

2.3. Tools to manipulate objects containing EtBr:

Laboratory instruments related to EtBr should be kept in separate places and clearly marked.

3. EtBr treatment methods currently in use in the world:

3.1.Use commercially available filter kit to remove EtBr in wastewater.

The filter agent is mainly activated carbon, the wastewater after filtration can be discharged into the sewer.

The commercially available filter kits are supplied from BIO 101, which is mainly activated carbon in the form of tea bags, each bag can absorb 10mg of EtBr, a kit of 50 bags should be able to absorb 500mg of EtBr from solution. Translate. Coal bags after treatment should be discarded as toxic chemical waste.

3.2. Discard solutions with low concentrations (less than 100 mg/ml).

3.2.1. Option 1 (Lunn and Sanrone 1987):

+ Add 2.9g Amberlite – XAD – 16 per 100ml of treatment solution.

Store the solution for 12 h at room temperature with occasional shaking.

+ Filter the solution through Whatman filter paper No.1. Discard the filtrate down the drain.

+ Put filter paper and Amberlite in a plastic bag, seal, discard.

3.2.2. Option 2 (Bensaude 1988).

+ Add 300mg of activated carbon powder for every 100ml of solution to be treated.

Leave the mixture at room temperature, shaking occasionally.

+ Filter the mixture through Whatman filter paper No.1. Discard the filtrate down the drain.

+ Put filter paper and Amberlite in a plastic bag, seal, discard.

Treatment for solutions with high concentrations (>= 0.5mg/ml) (Lunn and Sansone 1987).

– Step 1: Add enough water to lower the EtBr concentration to 0.5mg/ml, or less (done in a fume hood).

– Step 2: Add 20ml hypophosphorous acid 5% and 12ml 0.5mol Sodium nitrite, mix carefully. (Hypophosphorous is usually taken out at 50% concentration then diluted 10 times to get 5% just before use).

– Step 3: Prepare sodium nitrite: Weigh 3.45g sodium nitrite, then dissolve in water, bring to a volume of 100ml (done in a fume hood).

– Step 4: After incubation for 24 hours at room temperature, bring the pH to about 5-9 with sodium bicarbonate. Then pour the liquid down the drain.

* Do not use hypochloride to treat EtBr. Because treatment with bleach can produce more potent mutagenic products and the residual EtBr after treatment is 20%.

Surface decontamination due to EtBr.

– Wipe off spilled liquid with absorbent paper. Put garbage in a plastic bag, make a seal and keep it in a safe place. Items contaminated with EtBr should be kept separate and marked for identification.

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