Pairs of genomic insertions made with elements carrying any one of several frequently used rare restriction sites allow physical purification of insertion delimited genes. However, native rare restriction sites can, either by causing (i) fragmentation of targeted intervals or (ii) generation of additional fragments that overlap electrophoretically with targeted ones, place severe limitations on this approach. We present a series of Escherichia coli mini-Tn10 insertions containing the rare-cutting polylinker 2 (RCP2) of rare restriction sites, which includes the 18-base-pair I-SceI site (absent from native E. coli sequences). Pulsed-field gel purification from RCP2 double insertion mutants of both an I-SceI fragment from strain K-12 (containing approximately 90-95 min) and an allelic I-SceI fragment from a pathogenic strain is demonstrated. The complete series of RCP2 insertions, containing different antibiotic resistances at intervals of approximately 35 kb in prototype K-12 strain MG1655, allows rapid purification of the genes from any E. coli chromosomal interval as an isolated I-SceI fragment.