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Figure 4 | BMC Biochemistry

Figure 4

From: Bifunctional TaqII restriction endonuclease: redefining the prototype DNA recognition site and establishing the Fidelity Index for partial cleaving

Figure 4

Determination 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.

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