Figure 8

Digestion of highest complexity genomic DNA ( Equus caballus ) with TaqII/SIN/DMSO for BAC library construction. TaqII affinity star cleaved 1 μg horse liver DNA was electrophoresed on 1.5% agarose/TBE gel. Cleavage was carried out as described in Methods. (A) Partial digestion controlled by serial enzyme dilutions. Lane M1, Fermentas 100 bp DNA Ladder (selected bands marked); lane M2, Fermentas 1 kb DNA Ladder (selected bands marked); Lane K, undigested genomic DNA; lanes 1–4, DNA digested with 4 μg (32 pmols) of TaqII (Methods): lane 1, without SIN and DMSO; lane 2, with SIN, no DMSO; lane 3, no SIN, 20% DMSO; lane 4, with SIN, 20% DMSO; lanes 5–14, DNA digested with twofold dilutions of TaqII for 3 h at 65°C in the presence of SIN/DMSO: lane 5, 5 μg; lane 6, 2.5 μg; lane 7, 1.25 μg; lane 8, 0.63 μg; lane 9, 0.31 μg; lane 10, 0.16 μg; lane 11, 0.08 μg; lane 12, 0.04 μg; lane 13, 0.02 μg; lane 14, 0.01 μg. (B) Partial digestion controlled by reaction duration. Lane M1, Fermentas 100 bp DNA Ladder (selected bands marked); lane M2, Fermentas 1 kb DNA Ladder (selected bands marked); DNA digested with 5 μg (40 pmols) of TaqII at 65°C in the presence of SIN/DMSO (Methods): lane 1, 16 h; lane 2, 8 h; lane 3, 4 h; lane 4, 2 h; lane 5, 1 h; lane 6, 30 min; lane 7, 15 min; lane 8, 7.5 min; lane 9, 3.25 min; lane 10, 1.62 min; lane 11, 0.81 min.