用组合引物PCR法简便快速筛选阳性正向克隆

A Simplified Method for Quick Screening Positive Clones and Foreign DNA Orientation by Grouping Primers PCR

PCR扩增的小片段DNA用T载体连接,转化受体菌后观察不到典型的蓝/白斑。用插入片段的特异性引物对菌落进行PCR来筛选阳性正向重组子,测序鉴定结果吻合率达100%,说明当利用颜色特性挑选阳性克隆成为困难时,菌落PCR技术是一种简单、快速、可靠的筛选方法。

A typical color identification of blue/white colonies for recognition negative and positive recombinants can not be observed after small fragments of DNA being cloned into T- vector. Using specific primers of insert fragments, we amplified colonies obtained from sub-clone into E.Coli. The result of the sequencing of the positive recombinants selected by colony PCR shows that all the positive recombinants contain inserted fragments and Foreign DNA Orientation. So colony PCR technique is a simple, rapid and reliable screening method when it is hard to select the positive colonies by color characteristics.