Factors affecting quantitation of DNA bands in gels using a charge-coupleddevice imaging system

Quantitation of DNA bands separated in polyacrylamide or agarose gels was t ested under a variety of conditions to examine key factors contributing to the ability to obtain quantitative data. Variations tested included compari son of different fluorescent stains (ethidium bromide and GelStar stain), a nd variations of other parameters relating to the electrophoretic separatio n, e.g. gel and sample geometry, mode of staining, and mode of excitation o f 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. Variatio ns in separation and detection could lead to relatively large changes in fl uorescent signals seen for similar amounts of DNA.