Purification of Plasmid DNA

After the initial characterization, it is possible to purify further some or all of the plasmid DNAs by RNase digestion and extraction with organic solvents. This further purified DNA is suitable for techniques such as DNA sequencing, subcloning or the production of gene probes. In order to purify plasmid DNA after the isolation process, any residual RNA and contaminating protein are removed. This purification step involves two main steps, which are, first, removing residual RNA by using RNase in order to digest RNA and, second, extract contaminating protein using organic solvents, phenol-chloroform.

Rapid Boiling Method for Plasmid DNA Isolation

In the previous post, it had already explained about how to extract plasmid DNA by using alkaline lysis method. In this particular post, it will be explained the other method to extract plasmid DNA instead of using alkaline lysis method. It is Rapid Boiling method, an alternative to alkaline lysis method that was developed by Holmes and Quigley. Here, the cells are lysed partially allowing plasmids to escape, whereas the bacterial chromosomal DNA remains trapped in the cell debris. High temperature is then used to denature the chromosomal DNA, after which reannealing allows the plasmids to reassociate. Centrifugation removes the chromosomal DNA along with the cell debris, leaving the plasmid in suspension, from where it is recovered by isopropanol precipitation.

Plasmid DNA Extraction Using Alkaline Lysis Method

Plasmids can be isolated by a variety of methods many of which rely on the differential denaturation and reannealing of plasmid DNA compared to chromosomal DNA. One commonly used technique developed by Birnboim and Doly involves alkaline lysis. This method essentially relies on bacterial lysis by sodium hydroxide and sodium dodecyl sulfate (SDS), followed by neutralization with a high concentration of low-pH potassium acetate. This gives selective precipitation of the bacterial chromosomal DNA and other highmolecular-weight cellular components. The plasmid DNA remains in suspension and is precipitated with isopropanol.