Figure 2.

Both NTS are cleaved in the SEB product. (A) The products of a transposition reaction using a supercoiled plasmid substrate (pRC650) were digested within the plasmid backbone with the restriction enzyme BsaHI, dephosphorylated and 5′-labeled with γ-³²P-ATP and PNK. Products were separated on a native agarose gel and purified. The SEB products were tested for the presence or absence of single-strand nicks at the opposite end. This assay cannot distinguish if a single-strand nick is located on the NTS or on the TS. However, since the NTS is always cleaved first at a given transposon end ((22) and Supplementary Figure S1), a single-strand nick must be on the NTS. The unreacted substrate and backbone (BB) fragments were also gel purified and used as molecular markers. (B) The gel-purified SEB products (SEB-Right and SEB-Left), the unreacted full-length substrate and the backbone fragments (BB-Right and BB-Left) were analyzed by denaturing agarose gel electrophoresis. The gel was dried and recorded by autoradiography.