TN5MAP, A TRANSPOSON FOR THE RAPID MAPPING OF RESTRICTION SITES IN PLASMIDS

A transposon was constructed allowing the rapid restriction mapping of plasmids. This transposon, Tn5Map, contains a cleavage site for the I -SceI endonuclease which recognizes an 18-mer. After in vivo transposi tion of Tn5Map into the plasmid of interest, the plasmid is isolated a nd linearized with I-SceI. Splinkers labelled with digoxygenin and com plementary to the left and right end of the linearized molecule are ad ded and ligated. After partial digestion of the splinkered molecules w ith the restriction enzyme of interest, separation of the cleavage pro ducts in an agarose gel, and Southern transfer, the labelled fragments are visualized by the addition of the chemiluminescent substrate AMPP D and alkaline phosphatase. The restriction map can be directly read f rom the bottom to the top of the gel.