Figure 3

Analysis of cosmid knots by two-dimensional gel electrophoresis. DNA extracted from P4 vir1 del22 viral particles, which were amplified in bacteria that harboured P4cos-5, was electrophoresed in a gel slab of 0.4% agarose equilibrated with TBE buffer. The first gel dimension (top to bottom) was at 0.8 V/cm for 36 h at room temperature. The second gel dimension (left to right) was done in the same electrophoresis buffer at 3.4 V/cm for 4 h at room temperature. (a) Ethidium bromide staining of the gel. (b) The gel blotted to a nylon membrane and probed for P4cos-5. (c) Scheme showing the gel positions of linear molecules (L), unknotted circles (C), and knotted circles (K) of P4 DNA (white) and P4cos-5 (black). Dimeric P4cos-5 molecules (2 × 4.7 Kb) would ran nearly as P4 DNA (10 kb).