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Representative gel of ligation reactions run under the optimum conditions using the T2 template. The optimum reaction conditions are 30 mM Tris–HCl (pH 7.5), 10 mM DTT, 3 mM MgCl2, 10 µM ATP, 20% DMSO, 2.5 pmol acceptor molecule, 70 fmol donor molecule, 3.125 pmol T2 template and 3 U T4 DNA ligase at 30°C for 18 h. P represents product, I represents adenylated intermediate and S represents starting material. See Figure 2 for a description of the individual reactions.
