원저 ( Original articles ) ; Blunt End Ligation 의 최적반응에 관한 연구

To determine the optimal condition of blunt-ended DNA ligation for the purpose of the efficient cloning, blunt-ended vector and insert DNA fragments were ligated under the various conditions of buffer, temperature, reaction time, and the molar ratio of vector to insert DNA. The efficiency of ligation was monitored by both agarose gel electrophoresis and the transforming frequency of genetic markers in plasmid pRSK 116 in vivo. The results showed that the optimal incubation temperature and the concentration of ATP were 20-25℃ and 0.5 mM, respectively. Spermidine (1 mM) and polyethylene glycol (3%) stimulated blunt-end ligation, but more than 10 mM of spermidine and 2 mM of ATP completely inhibited the reaction. Higher concentration of salt also inhibited blunt-end ligation. According to the transforming frequency assay, the optimal molar ratio of vector to insert DNA was 1, which was different from that of sticky-end ligation. However, the variation of pH (6.6-8.2) and bovine serum albumin concentration (0-500 ㎍/㎖) showed little influence on the ligation.