The effect of DNA concentration on mobility I pulsed field gel electrophoresis

Abstract

The effect of DNA concentration on pulsed field gel electrophoretic mobility was inserts at 8-800 μg/ml and digested to completion with Not I. An eigth of each 100 μl insert was used to rpoduce DNA loads of 0.1 to 10 μg per lane. The mobility of single copy restriction fragments, as detected by hybridization, was ;largely concentration independent when DNA concentrations were 80 μg/ml or less. However, at DNA concentrations of 200 μg/ml and greater, dramatic effects of DNBA concentration are evident. In the worstcase, at 800 μg/ml.,the apparent size of a DNA fragment is almost 2.5 times its true size. At constant DNA concentrations, increasing the DNA mass loads by loading larger insert slices had no further effect on DNA electrophoretic mobility, although the bands were broader for bigger insert slices. Thus, for precise and accurate sizing in pulsed field gel electrophoresis the DNA concentration in agarose inserts should not be greater than 80 μg/ml (107 diploid human cells/ml agarose insert). © 1992 IRL Press at Oxford University Press.