Transposon insertion site identification by IPCR.

Cells were grown in LB medium at 37°C for 6 h. DNA was isolated, and 6 μg was digested with 2 U/μg DNA of the restriction endonuclease Sau3AI (NEB). Ligation with 400 U of T4 DNA ligase (NEB) was performed at room temperature for 2 h with 1.5 μg of the digested DNA. Inverse PCR (IPCR) was performed with 0.5 μl of ligated DNA using oligonucleotides 6212 and 6420 (or 7195 and 7196 in the case of the TnFLXPhyi series) in a standard PCR program with a 4-min extension time using a Mastercycler thermocycler (Eppendorf). Phusion DNA polymerase (NEB) was used for PCR amplification. The amplified fragment was analyzed by Sanger sequencing (IDT) using the universal sequencing oligonucleotide 6224.