To improve the ligation efficiency and effectiveness of exonuclease digestion, certain modifications were undertaken in their established protocols . In self-annealing precursor 1 used in this work, the 5-OH termini of 1 at their folded state were at a nick. As a result, its phosphorylation could be as much as 30-fold less effective than a protruding ssDNA 5-OH termini . Similarly, phosphorylation at a 5-OH termini present at an overhang in the cohesive end ligation precursors may not be as efficient as a linear protruding ssDNA 5-OH termini. Hence, a consecutive denaturation (95C heating for 5 min)rapid cooling at the ice steps (combinedly referred to as "snap-cooling") for removal of the secondary structure followed by a higher yield phosphorylation procedure consisting of spermidine and DTT additives was adopted for all the three ligation methods . As mentioned earlier, there are prior literature reports on cDNA preparation by either no digestion , exclusive Exo I or III alone , and combined (dual) Exo I/III digestion in various buffers without explaining the effectiveness of each (44) (Figure ). We hypothesized that the effectiveness of these exonuclease digestion steps (or the lack of it) in removing unligated precursors and splint would correlate with the attenuation of the NSA. Hence, the ligation was followed by various combinatorial digestion steps that are listed in Table (C1 -C9). Some of these (no digestion (C4), single enzyme Exo I/III digestion (C1 and C2), or combined Exo I and III digestion in ligase buffer (C3)) are adopted from prior literature reports (Figure ). The others (C5 -C9) are derived from our serendipitous finding that dual Exo I and III digestion in Exo III buffer alone displayed superior performance than combined Exo I and III buffer (below). In addition, this may have led to the discovery of a possible beneficial role of the Mg 2+ supplement (below). The concentrations of ligated substrates were adjusted between 0.8 -1.6 µM in exonuclease digestion.