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.