With these pilot observations in dual digestion, the experiments were replicated to see the overall quantitative trends as an effect of buffer compositions and Mg 2+ concentrations (conditions C5 -C9, Table ). A quantitative summary of these experiments in terms of end-RFU and NNSA has been provided in Table . Among the conditions, C5 utilized Exo III buffer alone in 1X concentration with final Mg 2+ concentration at 10 mM. Conditions C6 and C7 had 3X Exo III buffer (therefore having 33 mM Mg 2+ concentration) and 1X Exo III buffer with supplemented Mg 2+ (total Mg 2+ concentrations at 33 mM), respectively. In contrast, condition C8 had the presence of combined Exo I and III buffers in their 1X concentrations (total final Mg 2+ 16 mM). Condition C9, aiming to investigate the role of enhanced Mg 2+ to C8, was supplemented with Mg 2+ to a final concentration of 29 mM. For both selfannealing and cohesive end ligations, the NSA-mitigating beneficial role of digestion in Exo III buffer alone and higher Mg 2+ concentration (conditions C5 -C7) over combined Exo I and III buffer usage (conditions C8 and C9) were prominently visible (Figure and C, Figure -E and Figure ). This observation was in concurrence with that in the pilot studies involving dual digestion (above). In fact, the NSA for LRCA conditions following C5 -C7 digestions in self-annealing ligated cDNA 1C was almost negligible and 10 -15-fold lesser (for NNSA) than C8 -C9 digestions involving combined Exo I and III buffers (Figure , Figure ). As a result, it led to a completely flat baseline when cDNA 1C alone (i.e., without any primer) from C5 -C7 digestions was subjected to an LRCA amplification mix (Figure ).