Carr & Griffiths 1987

Preparative density-gradient ultracentrifugation of DNA
(SM Carr & OM Griffiths.1987. Biochem Genet 25:385-390)

    Under high centrifugal force, a solution of cesium chloride (CsCl) molecules will dissociate, and the heavy Cs+ atoms will be forced  towards the outer end of the tube, thus forming a shallow density gradient. DNA molecules placed in this gradient will migrate to the point where they have the same density as the gradient (the neutral buoyancy or isopycnic point). The gradient is sufficient to separate types of DNA with slight differences in density due to differing [G+C] content, or physical form (e.g., linear versus circular molecules). 

     In the experiment above, after centrifugation for 10 hrs at 100,000 rpm (450,000 x g), two distinct bands, corresponding to sheared linear nuclear DNA above and circular mitochondrial DNA below, are visible under ultraviolet light. The DNA has been mixed with the intercalating dye ethidium bromide, which enhances the density difference between the two forms and causes the DNA to fluoresce. The separate bands are collected by poking a hole in the bottom of the tube. The intact mtDNA is available for further biological analysis.


All text material © 2009 by Steven M. Carr