In addition to the buffer combination, a possible favorable effect of greater Mg 2+ concentration (33 mM over default 10 mM for standard 1X Exo III buffer) was noticeable for both splint-padlock and cohesive end ligations (Figure and) but was insignificant again for self-annealing ligation due to its low NSA RFU (Figure ). This was noticed when either Exo III buffer was present in 3X concentration (with final Mg 2+ at 33 mM) or with Exo III buffer in 1X concentration but with supplemented Mg 2+ (final Mg 2+ at 33 mM, Figure and). In fact, the beneficial effect of higher Mg 2+ concentration seemed to be also present for dual digestion even in combined Exo I and III buffers for splint-padlock and cohesive end ligated cDNAs 4C and 11C (Figure and), showing lower NSA. Next, we tested the effect of an increasingly greater concentration of Mg 2+ supplement (10, 22, 33, and 44 mM) with primer-assisted LRCA. It was noted that the higher limit of Mg 2+ concentration seemed to be 33 mM for self-annealing ligated cDNA 1C (Figure ). At 44 mM, the amplification RFU started to decrease when the Mg 2+ supplement was either added externally or present as part of the Exo III buffer.