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Friday, June 14, 2013

Large Scale Restriction Enzyme Digestion of DNA

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 Before adding the enzyme,  a sample (1ul)  was withdrawn.  After incubation for 30 minutes at 37 degrees, another sample (1uL)  was withdrawn from the reaction.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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