Figure 1.

Standardization of in vivo (OP)₂Cu probing. (A) Single hit conditions. Aliquots of 4.0 ml of culture of E.coli DH10B cells harboring plasmid pLW4 were subjected to treatment with 200 µl of increasing concentrations of mixture of OP and copper sulfate (lane 2, 5 mM OP/0.375 mM CuSO₄; lane 3, 80 mM OP/6 mM CuSO₄; lane 4, 160 mM OP/12 mM CuSO₄; lane 5, 400 mM OP/30 mM CuSO₄) and 200 µl of 580 mM 3-MPA. The reaction was terminated after 1 min by the addition of 200 µl of 0.1 M neocuproine to the reaction mix as described in Materials and Methods. Primer extension products of the plasmid from untreated culture (C) and near single hit conditions (S) are shown in lanes 1 and 2, respectively. G, A, T and C refer to sequencing lanes with the same primer. (B) Time and concentration dependence. In vivo (OP)₂Cu reaction was carried out with cells containing plasmid pLW4 alone (lanes 1, 3, 6 and 8) or in combination with pVN184 (lanes 2, 4, 7 and 9) for different time points as indicated in the figure. Lanes 1–4 and 6–9 were treated with 200 µl of a mixture of 20 mM OP/1.5 mM CuSO₄ and 40 mM OP/3 mM CuSO₄, respectively, and 200 µl of 580 mM 3-MPA as described in Materials and Methods. Primer extension product of plasmid DNA isolated from cells treated with CuSO₄ alone is shown in lane 5 (C).