4-1.3.3.1.2(b). Ethidium bromide-cesium chloride density gradient centrifugation
• Density gradient centrifugation can separate DNA, RNA and protein. It is a very efficient method for obtaining pure plasmid DNA.
• A density gradient is produced by centrifuging a solution of cesium chloride at a very high speed which pulls the CsCl ions towards the bottom. This process is referred as isopycnic centrifugation.
• The DNA migrates to the point at which it has density similar to that of CsCI i.e.1.7 g/cm3 in the gradient.
• In contrast, protein molecules having lower buoyant densities float at the top of the tube whereas RNA gets pelleted at the bottom.
Density gradient centrifugation in the presence of ethidium bromide (EtBr) can be used to separate supercoiled DNA from non-super coiled molecules. Ethidium bromide is an intercalating dye that binds to DNA molecules causing partial unwinding of the double helix. Supercoiled DNA have very little freedom to unwind due to absence of free ends and bind to a limited amount of EtBr resulting in very less decrease in buoyant density (0.085 g/cm3 ) than that of linear DNA (0.125 g/cm3 ). As a result, they form a distinctb and separated from the linear bacterial DNA. The EtBr bound to DNA is then extracted by n-butanol and the CsCl is removed by dialysis.
4-1.4. Isolation and Purification of RNA
RNA (Ribonucleic acid) is a polymeric substance consisting of a long single-stranded chain of phosphate and ribose units with the nitrogen bases adenine, guanine, cytosine and uracil bonded to the ribose sugar present in living cells and many viruses. The steps for preparation of RNA involve homogenization, phase separation, RNA precipitation, washing and re-dissolving RNA.
The method for isolation and purification of RNA are as follows-
1. Organic extraction method
2. Filter-based, spin basket formats
3. Magnetic particle methods
4. Direct lysis method.