Figure 5
Extending the Fidelity Index to REases partially cleaving DNA: determination of minimum and maximum values for TaqII. (A) TaqII Fidelity Index under star inhibitory conditions. 0.3 μg (= 1.2-pmol GACCGA recognition sites) PCR(GACCGA) substrate was digested with decreasing amounts of TaqII for 1 h at 65°C in the following buffer: 40 mM Tris-HCl, pH 8.0, at 65°C, 10 mM (NH4)2SO4, 10 mM MgCl2, 1 mM DTT, BSA 100 μg/mL. The final glycerol concentration was 5%. Lane K, undigested PCR fragment; lane M, Sigma PCR 20-bp Low Ladder (selected bands marked); lanes 1-8, digested PCR fragment: lane 1: with 120 pmol TaqII; lane 2, 60 pmol; lane 3, 30 pmol; lane 4, 15 pmol; lane 5, 7.5 pmol; lane 6, 3.75 pmol; lane 7, with 1.87 pmol; lane 8, 0.94 pmol. (B) TaqII Fidelity Index under star stimulated conditions. 0.3 μg (= 1.2-pmol GACCGA recognition sites) PCR(GACCGA) substrate was digested with decreasing amounts of TaqII for 1 h at 65°C in the following buffer: 40 mM Tris-HCl, pH 6.0, at 65°C, 10 mM MgCl2, 1 mM DTT, BSA 100 μg/mL. The final glycerol concentration was 5%. Lane K, undigested PCR fragment; lane M, Sigma PCR 20-bp Low Ladder (selected bands marked); lanes 1-8, digested PCR fragment: lane 1: with 120 pmol TaqII; lane 2, 60 pmol; lane 3, 30 pmol; lane 4, 15 pmol; lane 5, 7.5 pmol; lane 6, 3.75 pmol; lane 7, 1.87 pmol; lane 8, 0.94 pmol.