Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy

Abstract

Quantitation of nucleic acids is a fundamental tool in molecular biology that requires accuracy, reliability, and the use of increasingly smaller sample volumes. This unit describes the traditional absorbance measurement at 260 nm and three more sensitive fluorescence techniques employing Hoechst 33258, ethidium bromide, and PicoGreen. The range of the assays covers 25 pg/ml to 50 µg/ml. Absorbance at 260 nm has an effective range from 1 to 50 µg/ml; Hoechst 33258 from 0.01 to 15 µg/ml; ethidium bromide from 0.1 to 10 µg/ml; and PicoGreen from 25 to 1000 pg/ml. Curr. Protoc. Mol. Biol . 93:A.3D.1‐A.3D.14. © 2011 by John Wiley & Sons, Inc.