Determination of proper transposon insertion. Confirmation of transposon insertions was performed by PCR for presence of transposon (ermG) (A). “Mwt” = molecular weight marker, “+” = positive control (gDNA from E. coli/pSAM_Bt); “-“= a negative control (P. gingivalis ATCC 33277). All other lanes contain amplicons from PCR of individual colonies of transformed P. gingivalis. Panels (B) and (C) show PCR of the same samples using primers for the bla and himar1c19a genes respectively that are present in the plasmid, but which should be lost with proper insertion of the transposon. These three panels are a combination of separate gels; all which were run using identical PCR gDNA template for each of the separate reactions. (D) Nested semi-random PCR for individual mutant sequencing preparation. PCR from individual colonies was performed using primers to Mariner transposon and random primers ARB1 and ARB2 (Additional file 6: Table S6). Two rounds of nested PCR were performed. Negative controls of wild-type P. gingivalis strain ATCC 33277 (Pg), template only (T) and primer only (P) lanes precede thirteen individual mutants.
Klein et al. BMC Genomics 2012 13:578 doi:10.1186/1471-2164-13-578