Quantitation of Nucleic Acids and Proteins

Reliable quantitation of nanogram and microgram amounts of DNA and RNA in solution is essential to researchers in molecular biology. Two methods for direct absorbance measurements at 260 nm are described—the first is a traditional cuvette-based method, and the second is a microvolume method that requires no cuvettes or capillaries. In addition, three fluorescence techniques using Hoechst 33258, ethidium bromide, and PicoGreen reagent are presented in this unit. These five procedures cover a range from 5 to 10 ng/ml DNA to 15,000 µg/ml DNA. Reliable quantitation of proteins is possible using several types of assays. UV spectroscopy is the simplest approach but is limited in sensitivity. More sensitive assays that use Coomassie blue binding, bicinchoninic acid (BCA), and the Lowry reaction are also described. All assays are prone to amino acid composition errors and interference from assay solution components. Flow charts and tables to help with appropriate method selection are included. Curr. Protoc. Essential Lab. Tech. 5:2.2.1-2.2.36. © 2011 by John Wiley & Sons, Inc. Keywords: nucleic acid; protein; quantitation; fluorescence; colorimetric; electrophoresis; standards