[108] Use of CsCl density gradient analysis for determining the guanine plus cytosine content of DNA

Publisher Summary This chapter investigates the use of CsCl density gradient analysis for determining the guanine plus cytosine content of DNA. The buoyant density of DNA in CsC1 increases linearly with its content of guanine plus cytosine (GC). Thermally denatured DNA and naturally occurring single-stranded DNA behave in a similar manner but are uniformly higher in density. The ease and reproducibility of buoyant density determinations provide a standard method for evaluating the composition and other attributes, for example, strandedness, molecular weight, and degree of homogeneity of DNA samples. As each centrifuge cell passes directly over the light beam, the light passes through the solution and out through the upper window, where it is slightly deflected in the ease of the three wedge windows. Thus, each beam emerges from a different cell. The beams can be observed by holding a white card between the camera lens and the 4-cell mask and looking at the card from the direction of the mirror when green light is used in the optical system. Alternatively, a photograph can be taken by exposing to ultraviolet (UV) light a film at this position. Light passing through the air spaces of the cells produces the smaller images, which are toward the back of the ultracentrifuge. The light passing through the CsCl gradient is refracted toward the front of the ultracentrifuge. At this position, these images are not in focus and do not yet correspond to the final photographs taken at the plate holder.