Figure 3.

Optimization of ligation reactions. (A and B) ATP and MgCl₂ concentration-dependent ligations using (c + _pc)/T2. The MgCl₂ concentration was first optimized under standard reaction conditions of 30 mM Tris–HCl (pH 7.5), 10 mM DTT, 1 mM ATP, 2.5 pmol acceptor molecule, 70 fmol donor molecule, 3.125 pmol template and 3 U T4 DNA ligase at 30°C for 18 h. An optimum concentration of 3 mM MgCl₂ was found, which was then used in the subsequent ATP-dependent assay. (C) DMSO-dependent ligation reaction using (c + _pc)/C2 in 3 mM MgCl₂ and 10 µM ATP. (D) Time-dependent ligation reaction using (c + _pc)/T2 in 3 mM MgCl₂, 10 µM ATP and 20% DMSO. In each case, product is represented by squares and intermediate by circles.