Experimental information for Ethidium Bromide

Easy, inexpensive to use.

Detects 2 ng/band dsDNA in agarose or acrylamide, including denaturing gels

Detects ~100 ng/band RNA in non-denaturing agarose gels

Greater dynamic range on the ImaGo system than Polaroid film

Rapid quantitative analysis using the Totallab software.

Excitation Maximum: 302nm

Emission Maximum: 615nm

Conditions

Post-Staining: Run gel. Stain for at least 20 minutes in 0.5 micrograms/ml EtBr in water, TE or TBE on a shaker.

Destain for 20 minutes in water.

Pre-casting : Cast gel using a final concentration of 0.5 micrograms/ml EtBr.

Run gel using 0.5 micrograms/ml EtBr in running buffer.

Destain for 20 minutes in water (optional) TE, TAE or water can be used to make up the 0.5 micrograms/ml EtBr stock solution.

Emission Filters: Ethidium Bromide filter

Binding: EtBr is a non-covalent intercalating dye.

Detection Limits: Detection of ssDNA and RNA is less sensitive than for dsDNA.

Exact figures for ssDNA are not currently available. Sensitivity will always be better in acrylamide gels than in agarose gels because acrylamide gels have lower background.

Sensitivity: 2 ng dsDNA/band, depending on fragment size. ~100 ng RNA/band in non-denaturing gels. Larger fragments will bind more EtBr.

Linear Range: ~1.5 orders of magnitude.

CAUTION: Use gloves due to mutagenicity!

Useful tips for EtBr

Binding efficiency and sensitivity of EtBr with ssDNA and RNA is less than with dsDNA.

EtBr and SYBR Green I compete for DNA binding. Staining first with EtBr, then with SYBR Green-I will give better results than with EtBr alone, but inferior to pure SYBR Green I staining.

For use in agarose and polyacrylamide gels. To stain denaturing gels, wash gel to remove urea before staining.

Longer stain times do not improve sensitivity. However, when dealing with highly concentrated, tightly packed bands in a high percentage gel, allow more time for the EtBr to penetrate further into the bands to improve visualization. Staining and/or destaining for less than 20 minutes is not advised.

Destaining longer than 20 minutes can decrease band signal strength. Pre-casting gels with EtBr can save time after electrophoresis, but may also alter migration of fragments. EtBr migration causes non-uniform background staining in pre-cast gels. Post-staining should be used for quantitative analysis.

Store dye solution covered with aluminum foil to prevent bleaching of fluorophore by overhead lighting.

Stain solution can be re-used when stored at 4 degrees C, but must be strengthened by addition of fresh EtBr stock solution to compensate for that taken out of solution by the staining of previous gels.

Diluted stain solution will keep up to one month at 4 degrees C. It is not advisable to do either restriction digests or blotting after EtBr-staining. Powder can be stored at room temperature but must be protected from light.

In aqueous form, the stain should be stored at 4 degrees C protected from light. Use gloves due to mutagenicity.