You can do a triple digest since dpnI is compatible with cutsmart. Just increase the total volume of the rxn to keep final glycerol concentration low. just keep in mind that even with dpnI digestion you might still get a plasmid background.

You can do a triple digest since dpnI is compatible with cutsmart. Just increase the total volume of the rxn to keep final glycerol concentration low. just keep in mind that even with dpnI digestion you might still get a plasmid background.

Juan Minoia

You can do as many cuts as you wish if you account for plasmid concentrations and buffer compatibility. In practice I have worked with 4 enzymes that all were optimal in various buffers (NEB Buffers). But the main difference is salt concentrations, so if you needed buffer 1, 2 ,3 and cutsmart. You would first digest in buffer 1 with enzyme A, then buffer 2 with enzyme B etc.