A small slice of the plug should be digested with the primary restriction enzyme because less enzyme is required and other slices of the plug can be subjected to restriction analysis with secondary or tertiary enzymes, according to the table below. E. coli species, Salmonella, and Shigella sonnei utilize XbaI as the primary enzyme and BlnI as the secondary enzyme.
Shigella flexneri are tested with NotI as the primary enzyme and XbaI as the secondary enzyme. The use of a secondary (or tertiary) enzyme is useful in situations where the PFGE patterns obtained with the primary enzyme from two or more isolates are indistinguishable.