Figure 3

PCR verification of mini-Tn 7 - pir insertion and plasmid copy number. A) PCR verification of pir⁺ and pir-116 mini-Tn7 insertions into att Tn7 of E. coli DH5α. Colony PCR with primers 2372 and 2373 (see Figure 2 for relative priming site locations) was conducted to confirm presence or absence of the mini-Tn7-pir⁺ and mini-Tn7-pir-116 insertions. The PCR reactions were analyzed by agarose gel electrophoresis. Expected fragment sizes are 678 bp for DH5α without a mini-Tn7 insertion and 2,539 bp for derivatives containing mini-Tn7-pir⁺ or mini-Tn7-pir-116 insertions. Lane M, Hi-Lo molecular size ladder from Minnesota Molecular (Minneapolis, MN) with the sizes of selected fragments indicated; Lane -, DH5α negative control (no insertion). B) Demonstration of pR6KT2 copy number in pir⁺ and pir-116 containing E. coli and S. enterica serovar Typhimurium strains. Plasmid DNA was purified from overnight cultures of E. coli DH5α and S. enterica serovar Typhiumurium 14028S containing either mini-Tn7-pir⁺ or mini-Tn7-pir-116. A 20 μl aliquot of each preparation was digested with Hin dIII to linearize the 5,953-bp plasmid and the samples were analyzed by agarose gel electrophoresis. Lane M contains the same molecular size ladder as shown in panel A and the sizes of selected fragments are indicated.