Density Gradient Centrifugation in Angle-head Rotors.

Preparative isopycnic density-gradient centrifugation is ordinarily done in high-speed swinging-bucket rotors such as the Spinco SW 39 and SW 25. In addition to requiring this special equipment, the technique is severely limited by the small number of samples and the low total-sample volume which may be used. In recent studies on methods for isolating small numbers of virus particles from tissues and for separating nucleic acids which differed in density, we required means for centrifuging many samples simultaneously. The possibility of making such separations in ordinary angle-head rotors, which are widely available and have both a large number of tubes and high capacities, was therefore investigated. This application of angle-head rotors depends on reorientation of the density gradient from vertical to horizontal during deceleration, which in effect replaces the mechanical transition of the tubes in the swingingbucket rotor. This approach appeared feasible since the same basic principle had been applied to the construction of hollow, cylindrical, reorienting gradient rotors (l), in which a density gradient formed at rest is reoriented radially during centrifugation and is subsequently recovered at rest after deceleration. In this paper, data on the isopycnic banding of calf thymus DNA and T3 bacteriophage in CsCl are presented showing: that good resolution can be obtained with angle-head rotors; that fluid translations during deceleration do not cause appreciable mixing of the gradient or sample zone; and that the density gradients formed approximate the calculated compositional density gradients at equilibrium.