What is the role of Cscl and ethidium bromide in plasmid purification?

Answer 1

The CsCl forms a gradient and the molecules migrate according to their density until they float at their individual isopycnic points (the point in the gradient that equals the buoyant density of the molecule). However, plasmid DNA and contaminating chromosome have about the same density and cannot be separated easily. This is rectified, however, by the addition of ethidium bromide.

Density is a function of AT/GC ratio, but it is also a function of conformation. For supercoiled DNA, there is more DNA per unit volume than for relaxed DNA. Intercalation of ethidium bromide into DNA causes the helix to unwind (negative supercoiling) and become more relaxed. However, negative supercoiling only relaxes the DNA to a point. Further unwinding induces supercoiling in the opposite direction. When the DNA is circular and the ends are connected, the plasmid "kinks up" into a very tight knot. Thus, ethidium bromide causes the plasmid density to be increased.