Ethidium Bromide (EtBr) revelation

Goal

Protocol

1. When the gel electrophoresis is finished, turn off the generator and put the gel in a plastic box.


2. Put the box in the EtBr area.



SAFETY AND USEFUL RECOMMANDATION
This box must not touch any surfaces/objects contaminated with EtBr, because the box is very likely to be used after for another electrophoresis meaning that it will leave the EtBr area. Thus, this box must be cleaned with water and Ethanol before being reused.



3. Wear nitrile gloves over latex gloves, also wear the full facial protection.



SAFETY AND USEFUL RECOMMANDATION

Latex gloves do not protect against ethidium bromide, because it goes easily through the latex. Which is why nitrile gloves are available in the laboratory. These gloves are more protective. However they are not completely protective. Thus it is recommended to wear them over the latex one. It is also recommended to prevent to have direct contact between the gloves and EtBr. The full face protection protects both from solution projections and UV irradiance.



4. Open the box and put the gel (with the spatula) in the box containing the EtBr solution.



SAFETY AND USEFUL RECOMMANDATION

The gel should be put in the box before the EtBr solution in order to avoid the formation of gaz and droplets. The EtBr workbench is potentially contaminated. Thus, each tool used in this place has to stay inside the delimited area (white and red marking on the floor). You have to properly prepare your tools so you don't need to leave the area once you've started. Reduce as much as possible the import of new tools in the contaminated area in order to decrease the risk of EtBr propagation.



5. Prepare the EtBr solution (15 µL in 50 mL of deionized water).



SAFETY AND USEFUL RECOMMANDATION

The stock solution of EtBr is stored directly under the fumehood where the manipulations are performed. The product should always be stored at the indicated place. In case of incident like a spillage, users should not panic. you should prevent the liquid from spreading any further (especially outside the fume hood). As the ethidium bromide is not a volatile compound there is only little risk of inhalation. If there is a spillage outside the extracting hood follow the same procedure as if it didn't leave the fume hood. In case of spillage: Prevent the solution from spreading by using paper towel as dams around the puddle. Absorb the liquid with paper towel (always from the outside toward the inside). Put everything in the EtBr garbage. Clean the spot with water and dry the place after. Clean twice with alcohol and put everything in the EtBr garbage. The EtBr stock bottle has to be tightly closed and kept vertical.



6. After 5-10 min, put the gel in the washing bath and then put it under the UV lamp.



SAFETY AND USEFUL RECOMMANDATION

The gel is soaked with diluted EtBr. You have wash to the gel in the wash bath before putting it on the UV lamp. In case the gel falls off the spatula during its manipulation, two cases are possible. If the gel can still be used, take the gel and put it on the UV lamp. Then clean the spot as explained before. If the gel cannot be used just throw it in the appropriate trash. It is also possible that liquid was projected on your clothes. If it is the case it is recommended to throw them in the EtBr garbage.



7. Take a photo.



SAFETY AND USEFUL RECOMMANDATION
The UV lamp is switched on people who do not have any UV protection must not be nearby. The person working on the computer must have the same individual protection as the manipulator, that is to say: lab coat, nitrile gloves and full face protection. The sleeves have to cover the wrists and the collar of the lab coat has to be closed. Your skin should not be exposed to UV irradiance.