摘要
介绍一种使用ｐＵＣ质粒做为载体进行平端ＰＣＲ产物连接的方法。首先将特异的ＰＣＲ扩增产物纯化，不经任何处理，与用ＳｍａⅠ内切酶消化的载体ｐＵＣ质粒进行连接，并在连接体系中加入ＳｍａⅠ内切酶，２３℃连接１８～２０小时。转化宿主菌后，挑选白色菌落进行重组鉴定。在ＰＣＲ产物＜５００ｂｐ的ＤＮＡ片段重组阳性率占转化菌白色菌落的７０％，而＞１０００ｂｐ的ＤＮＡ片段重组阳性率占转化白色菌落的１０～２０％。
A new method,simple but of high performance,for the ligation of pCR blunt product with pUC Vector was presented here. Aafter being purified,the specific PCR product,without any further processing , was ligated with pUC Vector digested by SmaI in a ligation buffer containing l.3μl of T4 ligase and 1μl of SmaI.This reaction persisted for l8-20 hours under 23℃.After transformation,white colonies were selected for identifying the recombinant plasmid.The rate of positive recombination was 70～80%in PCR products of <500 bp while it is l0 ～20%in those of>1000 bp from selected white colonies,So,this method for blunt product ligation was simple,rapid and more effective when compared with that used routinely.
出处
《天津医科大学学报》
1995年第4期20-21,共2页
Journal of Tianjin Medical University
