Figure 2: Bce95I activity assay and run-off sequencing to determine cut sites. | Scientific Reports

Figure 2: Bce95I activity assay and run-off sequencing to determine cut sites.

From: Expression and purification of the modification-dependent restriction enzyme BisI and its homologous enzymes

Figure 2

(A) Bce95I digestion of pBRFM, phage λ, XP12, and T4gt DNAs. Bce95I enzyme dilution factors are indicated on the top of each lane. ScaI, ScaI-linearized pBRFM; Fnu4HI, Fnu4HI-digested pBRFM (note: the plasmid is resistant to digestion due to methylation); pBR, Fnu4HI-digested pBR322; “--”, uncut DNA; 2-log, 2-log DNA ladder. (B) DNA run-off sequencing of the Bce95I cleavage site Gm5CTGC and Gm5CAG m5CCGC of pBRFM. The up arrow indicates the bottom strand (template) is cleaved. The extra A peak indicates a cut in the bottom strand template (indicated by ↑ arrow). The extra T peak indicates a cut in the top strand (indicated by ↓ arrow). The color-coded sequence traces are: A (green), T (red), C (blue), G (black). The extra A trace (or T on the opposite strand) was added at the end of the cleaved template by the Taq DNA polymerase (template-independent terminal nucleotide transferase activity).

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