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. 2000 Jul 15;14(14):1789–1796.

Figure 2.

Figure 2

Southern blot of pQC110 and pQC26-derived DNAs isolated from ZX7 transformants. (A) DNA isolated by treatment with proteinase K and SDS (Qin and Cohen 1998) from transformants receiving DraI-cleaved pQC110 DNA. DNAs were electrophoresed for 13 hr at 40 v in 0.5% agarose gel and probed with 32P-labled pQC110 DNA (lanes 4–17). Molecular lengths were calculated relative to HindIII-treated bacteriophage λ DNA (lane 1), a 1-kb DNA-size ladder (Life Technologies, Inc.) (lane 2), or covalently closed circular pQC110 DNA isolated from E. coli (lane 3). (B) Surviving replicons are linear plasmids. Lanes 4–7 (NT) show DNA isolated from 4 randomly selected transformants by proteinase K/SDS treatment. Aliquots of the same DNAs were treated with 100 units exonuclease III (lanes 8–11) or 10 units λ exonuclease (lanes 12–15) at 37°C for 4 hr and electrophoresed for 18 hr at 38 v in 0.5 % agarose gel. λ HindIII-treated DNA (lane 1), 1-kb DNA ladder (lane 2), and pQC110 DNA (from E. coli, lane 3) are molecular size markers. (C) Electrophoresis of pQC110-derived DNAs shown in A after treatment with NaOH and renaturation. Lane designations are as in A. (D) BamHI digestion of pQC110-derived DNAs from A. Lane 1 contains a 1-kb ladder. Lanes 2–15 correspond to DNAs in lanes 4–17 of A. The 8.5-kb and 5-kb DNA bands discussed in the text are indicated. (E) BamHI digestion of pQC110-derived DNAs following denaturation and renaturation. Lanes 2–16 correspond to DNAs in lanes 3–17 of A. (F) Effect of denaturation on migration of BamHI fragments containing putative palindrome apices of linear plasmids. Agarose gel analysis of inserts recovered from agarose gel following BamHI digestion of pQC143–pQC146. The banding position of DNAs dissolved in TE (lanes 2–5) or analyzed following treatment with NaOH and neutralization is shown in lanes 6–9. (G) Endonuclease analysis of BamHI fragments containing putative palindrome apices. DNAs were digested by the enzymes indicated and electrophoresed for 3 hr at 80 v on 1% agarose gel. Lanes 2, 4, 6, 8, and lanes 10, 12, 14, 16 correspond to lanes 2–5 from F. Lanes 3, 5, 7, 9, and lanes 11, 13, 15, 17 correspond to lanes 6–9 from F. (H) Effect of denaturation on migration of SacI-cleaved pQC26 DNA isolated from four transformants by adding proteinase K/SDS (lanes 3–7) or NaOH/SDS (lanes 8–12), and electrophoresed for 20 hr at 36 v in 0.5% agarose gel. Lane 1 (1-kb ladder) and 2 (pQC26, from E. coli) are markers.