The preparative digestion of DNA with restriction enzymes is a core technique in all Biotechnology labs. Here we describe a best practices approach. One of the key factors in obtaining complete digestion of plasmid DNA is the quality of the DNA. Its important that RF1 (or supercoiled ) plasmid is the predominant form. Presence of substantial amounts of RFIII is usually indicative of significant DNA nicking. Usually the most important factor is that the DNA is free from the reagents used to purify it. For example, even very small traces of Phenol and SDS can exert a significant inhibition of restriction enzyme activity. Elsewhere is this blog, we will describe a robust routine method of plasmid DNA purification.
In the example below, 20ug of PBR322 plasmid were incubated in a reaction (20uL) containing 1X NEB buffer 2 and 10 units of HindIII. Before adding the enzyme, a sample (1ul) was withdrawn. After incubation for 30 minutes at 37 degrees, another sample (1uL) was withdrawn from the reaction. The "before " and "after " samples were run on 1% agarose gel electrophorsis.
As can be seen from the picture, complete digestion was obtained. Subsequent work up of the DNA digest depends on its future utility and will be discussed in that context in other posts.
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