Use of preparative CsCl gradients to estimate DNA buoyant densities: Potential errors due to fractionation artifacts

Abstract

The equilibrium banding behavior of high molecular weight DNA has been examined after centrifugation in CsCl gradients on angle rotors in the Spinco preparative ultracentrifuge. DNAs of base composition ranging from 30% GC to 71% GC, from a variety of species, band in close agreement to a calculated curve relating density and radial distance in angle preparative gradients, but only if the DNA is sheared prior to centrifugation. Unsheared DNA, after bottom tube puneture and drop collection by conventional methods, displays a characteristic unsymmetrical profile and the fraction with the largest concentration of DNA appears in a much lower fraction number than expected. This artifact leads to misealculation of buoyant densities from preparative gradients and interferes with fractionation of DNA, as for instance in separating satellite bands. Collection of the gradient from the top reverses the direction of the profile asymmetry and shearing eliminates it entirely.