FIG. 5.

Detection of the repaired donor DNA after IS492 excision. (A) Plasmid preparation of pAG949 after agarose gel electrophoresis. The following forms of the plasmid are indicated to the right of the gel: open circular (O.C.), supercoiled (S.C.), and the repaired pAG949 after excision of IS492 (repaired donor). The DNA molecular size marker (lane M) is the Promega λ/H3 markers, with sizes indicated to the left. (B) PCR assay for the repaired donor DNA, using primers complementary to eps (lanes 1 to 5), and controls for addition of template to the reaction mixtures, using primers complementary to bla (lanes 6 to 10). Strains tested for the repaired donor DNA were HMS174(DE3) containing pCR2.1 (no IS492) (lanes 5 and 6), pAG949 (wild-type IS492) (lanes 4 and 7), pAG957 (IS492ΔmooV::cam) (lanes 3 and 8), pAG957 and pAG954 (IS492ΔmooV::cam plus mooV-his₆) (lanes 2 and 9), and pAG957 and pAG954 (inducing conditions [0.01 mM IPTG]) (lanes 1 and 10). The molecular size marker (lane M) is the New England Biolabs 100-bp marker with sizes (in base pairs) indicated to the left.