Factors affecting quantitation of DNA bands in gels using a charge-coupled device imaging system.
Quantitation of DNA bands separated in polyacrylamide or agarose gels was tested under a variety of conditions to examine key factors contributing to the ability to obtain quantitative data. Variations tested included comparison of different fluorescent stains (ethidium bromide and GelStar stain), and variations of other parameters relating to the electrophoretic separation, e.g. gel and sample geometry, mode of staining, and mode of excitation of stains. The results showed that linear results were seen for a similar 20-30-fold range of DNA concentrations with both stains tested and that it is critical to standardize separations to obtain consistent results. Variations in separation and detection could lead to relatively large changes in fluorescent signals seen for similar amounts of DNA.
White
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BioWhittaker Molecular Applications, Rockland, ME 04841, USA.
Wu
M
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D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004996:Ethidium; D005456:Fluorescent Dyes; D007091:Image Processing, Computer-Assisted; D015203:Reproducibility of Results
D005456:Fluorescent Dyes; D004247:DNA; D004996:Ethidium