5 Preparation and Electrophoresis of Plasmid DNA

Publisher Summary Plasmids consist of double-stranded DNA and are generally isolated as circular molecules in which each of the single strands is a covalently closed circle. Because the single strands are also wound round each other to give the double helix, they are inextricably linked together and the strands cannot escape from each other even if the forces that normally keep them together in the double helix are broken. Such links have been called “topological bonds” and require that both the single strands be covalently closed, because a single break in just one strand introduces a point of free rotation that allow the now discontinuous strand to wind off the other and escape. The physical characteristics that permit separation are the relatively small size of plasmids, their covalently closed circular structure, and the fact that they are not bound to other cellular components in a lysate. Chromosomal DNA is very large in Escherichia coli , for example, about 4700kb, so that lysis of the bacteria and manipulation of the lysate inevitably results in random shearing of the chromosomal molecules. In general, the size of the resulting fragments depends on the vigour with which the lysate has been handled.