Factors Affecting Quantification of Total DNA by UV Spectroscopy and PicoGreen Fluorescence

Published

August 26, 2009

Author(s)

Marcia J. Holden, Ross J. Haynes, Savelas A. Rabb, Neena Satija, Kristina Yang, Joe Blasic

Abstract

The amount of total DNA in a preparation extracted from tissues can be measured several ways, each method offering advantages and disadvantages. It is of interest for the sake of accuracy in quantitation to compare methodologies and determine if good correlation can be achieved. Different answers can be clues to the physical state of the DNA. In this study, we investigated the lack of correlation between absorbance and fluorescent (PicoGreen) measurements of the concentration of DNAs isolated from plant tissues. We found that quantitation based on absorbance measurements correlated with measurements based on phosphorus content, while PicoGreen-based quantitation did not. Further, we found evidence of the production of single-stranded DNA under conditions where the DNA was not fragmented into small pieces. Finally, we document the high sensitivity of the PicoGreen assays to the detergent known as CTAB (cetyldimethylethylammonium bromide). The CTAB-based method is highly popular for low cost DNA extraction with many published variations for plant and other tissues. Removal of residual CTAB is important for subsequent quantitation of DNA using PicoGreen.

Citation

Journal of Agricultural and Food Chemistry

Volume

57

Issue

16

Pub Type

Journals

Keywords

DNA quantitation, PicoGreen, absorbance, fluorescence, DNA isolation, single-stranded DNA, cetyldimethylethylammonium bromide, CTAB

Citation

Holden, M. , Haynes, R. , Rabb, S. , Satija, N. , Yang, K. and Blasic, J. (2009), Factors Affecting Quantification of Total DNA by UV Spectroscopy and PicoGreen Fluorescence, Journal of Agricultural and Food Chemistry (Accessed December 26, 2024)