Tn 5Map, a transposon for the rapid mapping of restriction sites in plasmids

A transposon was constructed allowing the rapid restriction mapping of plasmids. This transposon, Tn 5Map, contains a cleavage site for the I-SceI endonuclease which recognizes an 18-mer. After in vivo transposition of Tn5Map into the plasmid of interest, the plasmid is isolated and linearized with I-SceI. Splinkers labelled with digoxygenin and complementary to the left and right end of the linearized molecule are added and ligated. After partial digestion of the splinkered molecules with the restriction enzyme of interest, separation of the cleavage products in an agarose gel, and Southern transfer, the labelled fragments are visualized by the addition of the chemiluminescent substrate AMPPD and alkaline phosphatase. The restriction map can be directly read from the bottom to the top of the gel.