Analysis of mutant M. hyopneumoniae by PCR. M. hyopneumoniae strain 232 was transformed with pMHC9-1. PCR was performed on extracted total DNA from 15 individual transformants (lanes 1–15) for identification of the tetM gene (A) and the pGEM-T plasmid backbone (B). A further 11 transformants underwent a further two rounds of “colony purification”. Total DNA was extracted and linker PCR performed using two primer pairs at either end of the transposon (left and right) (C). The same 11 transformants were passaged 15 times in Friis medium without selection, and all subsequently retained their resistance to tetracycline. Linker PCR was repeated using the same two primer pairs (left and right). Control samples for PCR reactions included: “no template” control (NT); untransformed M. hyopneumoniae strain 232 (NC); plasmid DNA positive control (PC).