Figure 3
Optimization of TaqII REase reaction conditions. (A) The pH activity range of TaqII REase. 0.3 μg (= 1.2-pmol recognition sites) PCR(GACCGA) substrate was digested with 0.75 pmol TaqII in the pH range from 5.5 to 9.5 for 30 min. at 65°C. 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, pH 8.5; lane 8, 9.0; lane 9, 9.5. The intensity of the 48 bp band on the series of gels was measured using UnScan-It software and the percentage of relative enzymatic activity calculated. (B) The influence of ionic strength on TaqII REase activity. 0.3 μg (= 1.2-pmol GACCGA recognition sites) PCR(GACCGA) substrate was digested with 2.4 pmol TaqII in the ammonium sulphate concentration range from 0 to 70 mM for 30 min. at 65°C. Lane M, Sigma PCR 20-bp Low Ladder (selected bands marked); lane K, undigested PCR fragment; lanes 1-8, 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. The intensity of the 48 bp band was measured on a series of gels using UnScan-It software and the percentage of relative enzymatic activity calculated.