Cloning of λ DNA fragments into pUC19 vector to study the ligation efficiency of NdeI-digested pUC19 and HindIII-digested pUC19 by T4 DNA ligase

Ligation experiments were conducted to examine the ligation efficiencies of Hind IIIdigested pUC19 and Nde I digested pUC19 vectors by T4 DNA ligase. Hind III-digested pUC19 was shown to ligate more efficiently than Nde I-digested pUC19. Upon close examination, Hind III-digested pUC19 could also ligate intermolecularly and intramolecularly whereas Nde I-digested pUC19 can only ligate intermolecularly. Ligation of Hind III-digested pUC19 yielded monomeric and multi-meric, circular plasmid with varying degree of supercoiling. Ligation of Nde I-digested pUC19 only yielded multi-meric and possibly linear ligated fragments. Ligation temperature of 4, 16, and 22ºC showed that temperature did not significantly affect the ligation efficiency. In order to study the effect of plasmid size on ligation efficiency, cloning of pUC19 fragments of sizes ~3686, 4686, and 6686 kb was attempted. Specific regions from λ DNA were amplified by PCR and these 1000, 2000, and 4000 bp fragments were cloned into pUC19 vectors. However, clones could not be obtained due to time constraints and technical difficulties in the screening step. Improvements on the screening system may increase the likelihood of obtaining desirable clones.