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. 2004 Jul;72(7):4010–4022. doi: 10.1128/IAI.72.7.4010-4022.2004

FIG. 2.

FIG. 2.

Representative gels of control experiments testing specificity of the tags and sensitivity of the PCR amplification to detect the tags on the chromosome. (A) The tag2 plasposon does not cross-amplify with the other tag oligonucleotides. PCR was performed using the plasposon pTnMod-OTp-tag2 as a template. The primer STM-common was used with a different tag-specific primer in each reaction. Lane M: 1-kb ladder molecular weight markers; lane 1, distilled H2O template with tag2 primer (reaction negative control); lane 2, pTnMod-OTp′ lacking tag insertions with tag2 primer (template negative control). Lanes 3 to 34 were loaded with amplification products from the reactions containing pTnMod-OTp-tag2 template and each of the various tagn primers, beginning with lane 3 using tag1 until lane 34 using tag32. PCR amplification is specific for tag2 as indicated by the lack of PCR amplification in all lanes except lane 4. (B) Tag oligonucleotides can be detected from the B. cepacia chromosome by PCR after transposition. For each PCR amplification, the primer STM-common was used in combination with the indicated tagn-specific primer. Lane M: 1-kb ladder molecular weight markers; lane 1, pTnMod-OTp-tag1 with the tag1 primer (template positive control); lane 2, pTnMod-OTp′ with the tag1 primer (template negative control); lanes 3 to 6, PCR amplifications with the tag1 primer using chromosomal DNA from four independent K56-2::tag1 transposon mutants; lanes 7 to 10, PCR amplifications with the tag2 primer using chromosomal DNA from four independent K56-2::tag2 transposon mutants; lanes 11 and 12, PCR amplifications with the tag1 (lane 11) and tag2 (lane 12) primers using chromosomal DNA from a mixed culture containing K56-2::tag1 and K56-2::tag2 mutants.