Gel electrophoresis with Ethidium bromide staining updated: Aug 2014

Safety information/requirements for this procedure:

Chemicals used:

Chemical(s) Used Hazard Class Health Hazards Ethidium Bromide (ETBR) Health 1 Fire 1 Sazardous in case of ingestion. Slighly hazardous in case of skin contact (irritant), of eye contact (irritant), or inhalation TAE buffer (Tris, Glacial Health 0 fire 0 None Acetic acid, EDTA)

Personal protective equipment required for this activity:

Nitrile gloves are required during the handling of stock solutions of Ethidium bromide and gels containing dilute Ethidium bromide. Eye protection and long sleeve clothing are recommended.

Other precautions required for this activity:

None

Designated work are for this procedure: Electrophoresis gel station

Agarose concentrations vary depending on application. The most common concentrations for activities in this lab are 0.7% and 1.4%.

0.7% gels are used when separating large molecules such as genomic DNA or when a PCR product simply needs to be checked for amplification.

1.4% gels are used when separating small molecules such as RADseq libraries, or RFLP r PCR products that are close in size to one another.

Gel sizes and TAE volumes: • Small gel: 65 mL TAE (HOWEVER, add agarose as if you were making a 50 mL gel, this is because approximately 15mL of TAE will evaporate during the melting step) This correction only applies to the small gels. • Medium gel: 150mL TAE • Large gel: 250mL TAE

1ul of 10mg/ml stock ethidium bromide solution is added to every 100 ml of TAE buffer, however no less than 1ul of ethidium bromide should be added to a single gel.

To make an agarose gel: • Measure TAE and add to Erlenmeyer flask • Weigh appropriate amount of agarose and add to Erlenmeyer containing TAE • Microwave until all agarose has dissolved (solution should be clear) • Add appropriate amount of ethidium bromide • Swirl Erlenmeyer in ice bath to cool solution to ~55 C. • Pour into gel form

Electrophoresis voltage and time vary depending on application and arrangement of the wells. Typically 120 volts is adequate to separate PCR products or DNA, times vary depending on gel density, size of fragments being separated, and well arrangement; adjust accordingly.

All handling of Ethidium bromide must be done with gloved hands.

This applies to both stock solutions as well as gels containing diluted ethidium bromide.

All Ethidium bromide containing gels must be disposed of as hazardous waste.