Assay for the quantification of intact/fragmented genomic DNA
Section snippets
Reagents
Dimethyl sulfoxide (DMSO), Tris–HCl base, ethylenediaminetetraacetic acid (EDTA), and MnCl2 were obtained from Merck (Darmstadt, Germany). Quant-iT PicoGreen was obtained from Invitrogen–Molecular Probes (Eugene, OR, USA). Hoechst 33258 (bis-benzimide), DNA type I (from calf thymus), highly purified unsheared genomic DNA (from calf thymus), DNase I (from bovine pancreas), bovine serum albumin (BSA), and ethidium bromide were purchased from Sigma–Aldrich (St. Louis, MO, USA). DNA size markers
Results and discussion
Preliminary observations in our laboratory showed that the accurate quantification of dsDNA by the most commonly used Invitrogen–Molecular Probes protocols with the fluorescent dyes Hoechst and PicoGreen [20] depends strongly on its degree of fragmentation, whereas its UV absorbance (at 260 nm) is not affected. These observations are in contrast to the unconsidered effect of dsDNA size on the fluorescence of DNA–dye complexes in the protocols and in the relevant literature [15], [16], [17], [18]
Acknowledgments
This work was funded by the European Social Fund (ESF), Operational Program for Educational and Vocational Training II (EPEAEK II), and particularly the Program IRAKLEITOS. We thank George Kilias and Stephanos Martimianakis (Department of Biology, University of Patras) for their valuable assistance in the gel electrophoresis experiments.
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