The splint binding regions in 4C have been illustrated in Figure . The stability of longer splintpadlock complexes even under denaturing conditions was once again evident during PAGE analysis of the ligation (before exonuclease digestion) reaction (Figure ). As the splint length increased, the cDNA band migrated more slowly indicating continued association of the splint with the cDNA. This, along with earlier PAGE involving 38 nt long splint, indirectly implied that even gel purification might not successfully mitigate non-specific amplification for splintpadlock ligated cDNA.