Gel retardation assay with MlyI, PleI and their variants.
Conditions are described in the Figure 3 legend.
Lane 1 is the size standard of λ DNA
digested by HindIII, φ174
DNA digested by HaeIII. 0.25 pmol of the BssSI–BsrFI digested pNB1 was incubated with 1 µl
of crude cell extracts containing ∼0.3
pmol of wild-type MlyI (lane 2) and its variants
E380A (lane 3), D403A (lane 4), D418A (lane 5). Same DNA substrate
(0.25 pmol) was also incubated with 1 µl
of crude cell extracts containing ∼0.45
pmol of wild-type PleI and its variants D399A,
D414A, D427A, shown in lanes 7, 8, 9 and 10, respectively. Lanes
6 and 11 are the controls of plasmid DNA only.