Microvolume Quantitation of Nucleic Acids
Quantitation of DNA and RNA by absorbance and fluorescence spectroscopy has been a powerful tool in life sciences for decades. Classic methods of nucleic acid quantitation require the filling of devices, such as cuvettes and capillaries, with sample (traditional methodologies are described in APPENDIX 3D). Analysis of microvolume samples has become of paramount importance as more molecular biology techniques yield progressively smaller amounts of isolated sample and require accurate quantitation of nucleic acids with minimal consumption of sample. Advances in photonic technologies have resulted in a pioneering microvolume system that combines fiber optic technology with the inherent physical properties of the sample to dramatically reduce measurement volumes, removing the need for cuvettes and capillaries. Since the introduction of the first microvolume instrument, several new designs are now available, providing opportunities to measure nucleic acids using much smaller amounts of material. Altogether, these systems not only reduce measurement volume (as little as 0.5 to 2 µl), but also tend to be more efficient time-wise than traditional methods, making them useful even when sample is plentiful. The protocols in this unit are based on the most widely accepted microvolume systems and are intended as practical alternatives to traditional nucleic acid quantitation methodology. Curr. Protoc. Mol. Biol. 93:A.3J.1-A.3J.16. © 2011 by John Wiley & Sons, Inc.
Keywords: spectroscopy; DNA; RNA; quantitation; microvolume; absorbance; fluorescence
Table of Contents
- Basic Protocol: Microvolume Nucleic Acid Quantitation Using a Nanodrop Spectrophotometer
- Alternate Protocol 1: Microvolume Nucleic Acid Quantitation Using a Traditional Spectrophotometer and a Microcell Cuvette
- Alternate Protocol 2: High-Sensitivity Microvolume Nucleic Acid Quantitation Using a Nanodrop Fluorospectrometer
- Literature Cited
Basic Protocol: Microvolume Nucleic Acid Quantitation Using a Nanodrop Spectrophotometer
Alternate Protocol 1: Microvolume Nucleic Acid Quantitation Using a Traditional Spectrophotometer and a Microcell Cuvette
Alternate Protocol 2: High-Sensitivity Microvolume Nucleic Acid Quantitation Using a Nanodrop Fluorospectrometer
Figure A.3J.2 The shorter the path length, the higher the concentration that can be measured.
Figure A.3J.4 (A) Flattening of the sample droplet is indicative of an unconditioned optical pedestal. (B) Beading-up of the sample droplet is indicative of a properly conditioned optical pedestal.
Figure A.3J.5 A typical nucleic acid absorbance spectrum.
Figure A.3J.7 Detection limits and respective mass consumptions for a microvolume fluorospectrometer, a traditional cuvette-based fluorometer, and a fluorescence plate reader.
Figure A.3J.9 Spectral interpretation. (A) Typical nucleic acid sample absorbance spectrum. (B) Comparison of nucleic acid sample absorbance spectra with and without two common contaminants.
Figure A.3J.10 Several examples of reagents with absorption in the 220 to 240 nm range that are commonly used with nucleic acids.
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