The transgenic RescueMu lines were designed for and successfully used in our maize gene discovery project. The pBluescript-containing RescueMu transposon can be readily recovered by a procedure called plasmid rescue. Plasmid rescue is a technique for recovering bacterial plasmids from transgenic eukaryotic genomic DNA. Total maize DNA was first digested with restriction enzyme(s), ligated, and then transformed into E. coli cells. Colonies were recovered under selection against the antibiotic marker(s) in the transgene vector. Ampicillin or carbenicillin was used for RescueMu transgene recovery. The flanking genomic sequences at RescueMu insertion sites were simultaneously captured and then sequenced using RescueMu-readout primers. Genomic DNA from an individual plant or from pooled samples of up to ∼50 plants could be used in a single rescue. Because the majority of transgenic constructs currently used in flowering plants were made in the form of plasmids, this protocol could therefore be adapted by and useful to researchers involved in other transgenic work and be versatile for characterizing transgene loci.