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Abstract
NdeI digested fragments have a low ligation efficiency, and the cause is unknown. One possible explanation is the unavailability of NdeI digested ends to the ligase enzyme. Using PCR, a DNA fragment was created, which was the same sequence and size of pUC19 digested with NdeI and HindIII. The NdeI/HindIII digested fragment had slower mobility and appeared to be larger than its PCR counterpart when run on a polyacrylamide gel. To assess the accessibility of the 3’ ends of NdeI cuts, the 3’ recess was filled with a biotinylated dATP using Klenow enzyme. Only one 3’ end appeared to be available to the Klenow enzyme. However due to unequal retaining efficiency of the fragments to nitrocellulose, the labelling efficiency of each end of NdeI and HindIII remain unknown.