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Abstract
It is known that the rate of ligation for DNA digested with different restriction enzymes is variable. In order to further investigate this observation, pUC19 DNA was digested with one of seven different restriction enzymes to produce different DNA termini. All termini used in this study were blunt-ended either by restriction digest, by end filling, or excision of digested DNA with the Klenow fragment or T4 DNA polymerase. The resulting DNA was ligated with T4 DNA ligase and the effect of incubation time on the ligation efficiency was examined. Highest ligation efficiencies were observed at 0.5 hr whereas further incubation times lead to decreases in ligation efficiency. Additionally, Sma I, Sca I, and Xmn I digested blunt-ended DNA fragments displayed the highest ligation efficiencies in comparison to those fragments that underwent 5’ end filling or 3’ end excision to become blunt-ended. At 0.5 hr, the efficiency of ligation was greatest for Sma I digested sample and lowest for the Sal I and Sap I digested samples.