Microvolume Quantitation of Nucleic Acids

Philippe R. Desjardins1, Deborah S. Conklin1

1 Thermo Scientific NanoDrop Products, Wilmington, Delaware
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Appendix 3J
DOI:  10.1002/0471142727.mba03js93
Online Posting Date:  January, 2011
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: 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
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Microvolume Nucleic Acid Quantitation Using a Nanodrop Spectrophotometer

  Materials
  • Deionized water or appropriate buffer
  • Sample containing nucleic acids
  • NanoDrop 2000 or 2000c Spectrophotometer (Thermo Scientific NanoDrop Products, http://www.thermoscientific.com/nanodrop)
  • Laboratory wipes (e.g., Kimwipes)
  • Pipettor (0 to 2 µl)
  • Nuclease‐free, low‐retention pipet tips

Alternate Protocol 1: Microvolume Nucleic Acid Quantitation Using a Traditional Spectrophotometer and a Microcell Cuvette

  Materials
  • Sample containing nucleic acids
  • Microcell cuvette: TrayCell (Hellma, http://www.hellmausa.com/) or Optical Ultra LabelGuard Microliter cell (Implen, http://www.implen.de)
  • UV‐vis spectrophotometer (any standard cuvette spectrophotometer)
  • Pipettor (0 to 10 µl)
  • Nuclease‐free, low‐retention pipet tips
  • Laboratory wipes (e.g., Kimwipes)

Alternate Protocol 2: High‐Sensitivity Microvolume Nucleic Acid Quantitation Using a Nanodrop Fluorospectrometer

  Materials
  • Sample containing dsDNA
  • PicoGreen dsDNA Quantitation Kit (Invitrogen) including:
    • 20× TE buffer
    • dsDNA standards
    • PicoGreen dsDNA reagent
  • Nuclease‐free H 2O (e.g., Invitrogen)
  • Nuclease‐free, low‐retention pipet tips (e.g., Hamilton)
  • 1.5‐ml nuclease‐free amber microcentrifuge tubes or standard nuclease‐free microcentrifuge tubes wrapped in aluminum foil
  • NanoDrop 3300 Fluorospectrometer (Thermo Scientific NanoDrop Products, http://www.thermoscientific.com/nanodrop)
  • Laboratory wipes (e.g., Kimwipes)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Glasel, J.A. 1995. Validity of nucleic acid purities monitored by A260/A280 absorbance ratios. Biotechniques 18:62‐63.
   Ingle, J.D. Jr. and Crouch, S.R. 1988. Spectrochemical Analysis. XV +. Prentice Hall, Englewood Cliffs, N.J.
   Voolstra, C., Jungnickel, A., Borrmann, L., Kirchner, R., and Huber, A. 2006. Spectrophotometric Quantification of Nucleic Acids: LabelGuard Enables Photometric Quantification of Submicroliter Samples Using a Standard Photometer. Implen Applications Note, Munich, Germany.
   Wilfinger, W.W., Mackey, K., and Chomczynski, P. 1997. Effect of pH and Ionic Strength on the Spectrophotometric Assessment of Nucleic Acid Purity: BioTechniques 22:474‐481.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library