Destaining in water or 1 mM MgSO 4 may be required to achieve full sensitivity.Īs an alternative, ethidium may be included in the gel, but not the buffer. This method will detect approximately 5ng of DNA. Bands will appear bright orange on a faint orange background. Upon completion of the run, place gel in plastic wrap on a UV lightbox. Prepare enough buffer ( TBE or TAE) to fill the apparatus.(Stocks are generally 10 mg/ml, and require 5µl stock/100ml gel). Add EtBr to 0.5 µg/ml final concentration.Dissolve agarose in the buffer as per the standard protocol for preparing an agarose gel.Method I - Including Ethidium Bromide in the Gel and Buffer GEL PREPARATION HANDLE ONLY WITH GLOVES AND PROPER PRECAUTIONS. Another limitation is that the fluorescence of ethidium is quenched by polyacrylamide, reducing sensitivity by 10-20 fold in PAGE gels.ĭetection of DNA/RNA using Ethidium BromideĬAUTION: ETHIDIUM BROMIDE IS A POTENT MUTAGEN. Staining of denatured, ssDNA or RNA is relatively insensitive, requiring some 10 fold more nucleic acid for equivalent detection. It is important to note that ethidium staining is strongly enhanced by the double stranded structure of native DNA. Nonetheless, the sensitivity, simplicity ( the dye may be run in the gel with the DNA if desired, eliminating a separate staining/destaining process), and nondestructive nature of ethidium bromide staining have made it the standard stain for double-stranded DNA. Solutions must be handled with extreme caution and decontaminated prior to disposal. The major drawback to ethidium bromide is that it is a potent mutagen. It yields low background and a detection limit of 1-5 ng /band. The fluorescence of ethidium bromide in an aqueous solution is significantly lower than that of the interchelated dye.Įthidium bromide is a sensitive, easy stain for DNA. Ethidium re-emits this energy as yellow/orange light centered at 590 nm. Additionally, it can absorb energy from nucleotides excited by the absorbance of 260 nm radiation. Ethidium bromide possesses UV absorbance maxima at 300 and 360 nm. Ethidium bromide is a DNA interchelator, inserting itself into the spaces between the base pairs of the double helix. The most commonly used stain for detecting DNA/RNA is ethidium bromide. Fundamentals of Liquid Scintillation Countingīands in gels stained with ethidium bromide fluoresce under ultraviolet light.Applications of Liquid Scintillation Counting.Fundamental Principles of Electrophoresis.
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