Figure 2.
Representative gel of ligation reactions run under standard conditions using the T2 template. The standard reaction conditions are 30 mM Tris–HCl (pH 7.5), 10 mM DTT, 10 mM MgCl2, 1 mM ATP, 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. The reaction components are shown above each lane. The first lane contains a generic 25 base size control, the next four lanes are the four unreacted donor molecules and the next two lanes are the synthetic (a + pa) and (c + pa) size controls. The next four panels show the 16 possible reactions. The next three lanes show the ligation of (a + pa) without template, with template and treated with calf alkaline intestinal phosphatase after the reaction has gone to completion, and without ligase.