Next, we subjected the ligation reactions involving 16 -30 nt splints towards dual exonuclease digestion under C6 and C7 conditions (selected due to beneficial higher magnesium concentration) (Figures and). The successive reduction in splint length decreased NSA end-RFU and NNSA under both C6 and C7 conditions (Figure and 5C, Figure ). Due to low NSA under LRCA for both C6 and C7 conditions with 16 nt splint, they were further investigated for NSA during HRCA conditions. Accordingly, splint lengths 24 nt or higher were not further probed for HRCA as they already showed higher NSA under the LRCA conditions. For HRCA conditions, the NNSA for 16 nt splint ligated cDNA was less than 5% for both C6 and C7 conditions with near-zero end-RFU (Figure ). As a result, this produced an almost flat baseline for "cDNA alone" experiment in the representative amplification (Figure ). However, it was unclear from these experiments whether the lower splint length-based dissociation from cDNA (and accordingly lower NSA) was further aided by either the temperature (during snap-cooling) or exonuclease digestion or both. Overall, findings validated our hypothesis concerning thermodynamic stability (and its destabilization) of the splint-cDNA complex involving splint-length.