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Mapping of the SgrAI secondary sites on pBR322. Digestions were carried out in 40 μl of NEBuffer4 (buffer composition is listed in Materials and Methods) containing 2 μg of pBR322 DNA. Lanes: 1, uncut DNA; 2, DNA digested with 5 units of SgrAI for 1 h; 3, DNA digested with 50 units of SgrAI for 20 h; 4, BsrFI-cleaved DNA sequentially digested with 50 units of SgrAI for 20 h; 5, DNA digested with BsrFI; M, 0.1–10-kb DNA ladder.
