The buffer composition for Exo I is 67 mM glycine-KOH, 6.7 mM MgCl2, 10 mM mercaptoethanol pH 9.5 (25 C). On the other hand, Exo III and ligase buffer consisted of 10 mM bistris-propane-HCl, 10 mM MgCl2, 1 mM DTT enzyme at pH 7 (25 C) and 50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT at pH 7.5 (25 C), respectively. When Exo I and III buffers are mixed 1:1 (as in conditions C8 and C9), the net pH would shift towards the alkaline direction. In contrast, in either Exo III buffer alone (in conditions C5 -C7) or in ligase buffer (C3 condition), the pH was 7 -7.5. Therefore, the lesser effectiveness of dual exonuclease digestion in combined Exo I and III buffers could be due to a possible yet unknown functional inability of Exo III to perform in a pH higher than 7.5. Exo III, a 3-to 5-exonuclease specializes in digesting nicks (present in self-annealing substrate) and 3-recessive ends-containing sites (present in splint-padlock and cohesive end substrates). Therefore, its loss of functionality, in turn, affected digestion in all three self-annealing, splint-padlock, and cohesive end ligation that contained these sites, increasing the NNSA for C8 -C9 conditions in all three substrates. Apart from the stated pH-dependent loss of function, lower digestion performance might also stem from a yet unknown inhibitory effect from a component present in the Exo I buffer. The higher activity in increased Mg 2+ concentration was plausibly due to the fact that the ion is a cofactor in both these exonuclease enzymes . Overall, these data quantitatively demonstrated, for the first time (to the best of our knowledge), that dual Exo I and III enzymatic treatment of ligated substrates in Exo III buffer alone was more effective in reducing NSA than in combined Exo I and III buffers. This counterintuitive behaviour was most likely due to a yet unknown pH-modulated inhibition mechanism in the Exo III enzyme. Similarly, a possible beneficial effect of increased Mg 2+ presence towards mitigating NSA was observed. Although highly interesting, these observations were not mechanistically probed further in this study and would be investigated in future works.