Biochemical Techniques and Instrumentation

Agarose electrophoresis - Principle

This method of quantification is based on the ethidium bromide fluorescent staining of DNA. Ethidium bromide is a fluorescent dye, which intercalates between the stacked bases. The fluorescent yield of the dye-DNA complex is much greater than the unbound dye. UV irradiation at 254nm is absorbed by the DNA and transmitted to the dye and the bound dye itself absorbs radiation at 302nm and 366nm. This energy is retransmitted at 590nm, the reddish-orange region of the visible spectrum. In case of genomic DNA, the nucleic acids are electrophoretically separated on a 0.7-0.8% agarose gel containing ethidium bromide at a final concentration of 0.5 mg/ml. The quantity of DNA can be estimated by comparing the fluorescent yield of the samples with a series of standards, for instance, lambda ( λ ) DNA at varying known concentrations. This provides a very rapid and sensitive means of estimating the nucleic acid concentration. A large number of samples with as little as 1-5mg of DNA can be quantified. Besides quantification, it also provides the advantage of analyzing the quality of the DNA preparation. Native DNA, which migrates as a tight band of high molecular weight (>40 kb), presence of RNA, and degraded/sheared DNA, if any, can be visually identified on the gel.