Figure 4From: Bifunctional TaqII restriction endonuclease: redefining the prototype DNA recognition site and establishing the Fidelity Index for partial cleavingDetermination of reaction conditions minimizing and stimulating TaqII star activity. (A) Influence of pH on TaqII star activity. 0.3 μg (= 1.2-pmol GACCGA recognition sites) PCR(GACCGA) substrate was digested with 12 pmol TaqII in the pH range from 5.5 to 9.5 for 16 h at 65°C in the reaction buffer: 40 mM of the appropriate buffering agent, 10 mM MgCl2, 1 mM DTT, BSA 100 μg/mL. Lane M, Sigma PCR 20-bp Low Ladder (selected bands marked); lane K, undigested PCR fragment; lanes 1-9, digested PCR fragment: lane 1, at pH 5.5, lane 2, 6.0; lane 3, 6.5; lane 4, 7.0; lane 5, 7.5; lane 6, 8.0; lane 7, 8.5; lane 8, 9.0; lane 9, 9.5. (B) Influence of ionic strength (ammonium sulphate) on TaqII star activity. 0.3 μg (= 1.2-pmol GACCGA recognition sites) PCR(GACCGA) substrate was digested with 12 pmol TaqII in the ammonium sulphate concentration range from 0 to 70 mM for 16 h at 65°C in the following reaction buffer: 40 mM Tris-HCl, pH 8.0 at 65°C, 10 mM MgCl2, 1 mM DTT, BSA 100 μg/mL. Lane M, Sigma PCR 20-bp Low Ladder (selected bands marked); lane K, undigested PCR fragment; lanes 1-9, digested PCR fragment: lane 1, without (NH4)2SO4; lane 2, with 10 mM (NH4)2SO4; lane 3, 20 mM; lane 4, 30 mM; lane 5, 40 mM; lane 6, 50 mM; lane 7, 60 mM; lane 8, 70 mM.Back to article page