| Approved by: S. Thompson | SOP No. LBL09001 |
| Signed: | Effective from: |
| Date: | Last edited: |
This SOP deals with storage, handling, and disposal of Ethidium Bromide (EtBr). EtBr is used for visualisation of agarose gels. It is a powerful mutagen and must be handled with care.
The Biohackspace's EtBr stock is a solution created from 1 gram EtBr combined with 100 ml of TBE. The Biohackspace does not stock EtBr at any higher concentration, and in particular does not stock granular (powdered) pure EtBr.
This SOP must be consulted whenever EtBr is used in the lab and its procedures must be followed.
The EtBr user is responsible for his or her own safety as well as the safety of others in the lab. In general, the EtBr user should be very careful with any solutions of EtBr or solutions containing EtBr.
The lab has a designated Ethidium Bromide area. All work with EtBr must be performed in this area.
Gloves must be worn prior to any application involving EtBr, including opening vessels containing EtBr, adding EtBr to molten agarose, and disposal of EtBr-contaminated material.
The Ethidium Bromide area contains glassware (such as flasks) which may be used for EtBr-related procedures. Do not add anything containing EtBr to any other glassware.
EtBr solution is stored in a screw-top bottle. The bottle must remain closed until EtBr is required, and must be re-closed immediately after use.
EtBr may be added to molten agarose to make a gel suitable for visualisation.
- Transfer the EtBr bottle to the EtBr area. Ready a pipette tip.
- Remove the vessel containing molten agarose from the microwave.
- Wait until the temperature of the molten agarose is below 60 degrees C.
- Unscrew the EtBr bottle, pipette the required amount, immerse the pipette tip in the molten agarose, and add the EtBr.
- Close the EtBr bottle.
Note that the EtBr must be applied after the agarose has been heated. This is to prevent contamination of the microwave.
Solids -- including pipette tips, gloves, and gels -- contaminated with EtBr must be disposed in the the EtBr waste bin.
Liquids containing EtBr must be poured into the EtBr waste bin.
Any surfaces used during the procedure must be wiped with bleach.
EtBr glassware may be cleaned with hot water and detergent and returned to the Ethidium Bromide area.
In the case of a spillage of liquid containing EtBr the following procedure should be carried out:
- Absorb the spill with paper towel and dispose of the towel in the EtBr waste bin. Repeat the procedure if necessary to remove all liquid containing EtBr.
- Wearing UV-proof goggles, visualise the extent of the spill using 300nm UV light. EtBr glows orange under UV.
- Decontaminate the area by scrubbing thoroughly with bleach using paper towel. This paper towel may be disposed of via the general waste.
- Visualise the area under UV light again to ensure that all EtBr has been removed, and repeat the procedure if any remains.
If the spill covered a large area (for example, it covers multiple bench areas or spills onto the floor or equipment), if the spill involves more than 10ml of liquid EtBr stock, or if if you are not certain that the spill has been entirely decontaminated, you should notify the BSO immediately.
The EtBr waste bin must not be allowed to fill beyond half full before emptying.
The Armour method for EtBr deactivation is followed for deactivating the contents of the EtBr waste bin before emptying.
A labcoat, eye-protection and gloves must be worn during the following procedure.
- Move the bin upstairs and take it outside, so that any volatile substances produced during the bleaching portion of deactivation may disperse.
- Add household bleach to the bin until the total volume of liquid has doubled.
- Let sit for 2-3 days.
- Use sodium hydroxide to adjust pH to 4-9. Test using universal indicator strips.
- Decant the liquid into an upstairs drain, washing down with copious amounts of water.
- Securely double bag any solid waste and dispose of via the general waste.