S1 nuclease digestion of supercoiled DNA. Supercoiled recombinant plasmid containing the inverted repeat was treated with S1 nuclease in a reaction buffer containing 200 mM NaCl for 1 min (lane 1), 2 min (lane 2), or 5 min (lane 3) at 25°C followed by DraI digestion. DraI-digested, intact DNA (S1-untreated) was loaded onto lane 4. DNA size markers (New England BioLabs, Beverly, MA) were loaded onto lane M. The efficiency of specific S1-cutting was evaluated by comparing the intensity of the top and bottom DNA bands; ∼40% of the plasmid molecules were specifically cut at the inserted DNA region after 5 min of incubation.