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Title

CGE-laser induced fluorescence of double-stranded DNA fragments using GelGreen dye

AuthorsValdés, Alberto ; García-Cañas, Virginia ; Cifuentes, Alejandro
KeywordsDNA separation
Fluorescent DNA-staining dye
GelGreen
PCR
CGE-LIF
Issue Date2013
PublisherWiley-VCH
CitationELECTROPHORESIS 34(11): 1555-1562 (2013)
AbstractNowadays, new solutions focused on the replacement of reagents hazardous to human health are highly demanded in laboratories and Green Chemistry. In the present work, GelGreen, a new nonhazardous DNA staining reagent, has been assayed for the first time to analyze double-stranded DNA by CGE with LIF detection. The effect of GelGreen concentration on S/N ratio and migration time of a wide concentration range of standard DNA mixtures was evaluated. Under optimum GelGreen concentration in the sieving buffer efficient and sensitive separations of DNA fragments with sizes from 100-500 base pairs (bp) were obtained. A comparison in terms of resolution, time of analysis, LOD, LOQ, reproducibility, sizing performance, and cost of analysis was established between two optimized CGE-LIF protocols for DNA analysis, one based on the dye YOPRO-1 (typically used for CGE-LIF of DNA fragments) and another one using the new GelGreen. Analyses using YOPRO-1 were faster than those using GelGreen (ca. 31 min versus 34 min for the analysis of 100-500 bp DNA fragments). On the other side, sensitivity using GelGreen was twofold higher than that using YOPRO-1. The cost of analysis was significantly cheaper (ninefold) using GelGreen than with YOPRO-1. The resolution values and sizing performance were not significantly different between the two dyes (e.g. both dyes allowed the separation of fragments differing in only 2 bp in the 100-200 bp range). The usefulness of the separation method using GelGreen is demonstrated by the characterization of different amplicons obtained by PCR. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
URIhttp://hdl.handle.net/10261/99861
DOI10.1002/elps.201200624
Identifiersdoi: 10.1002/elps.201200624
issn: 0173-0835
e-issn: 1522-2683
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