单菌落PCR在发夹RNA重组克隆鉴定中的应用

Application of Individual Bacterial Colonies PCR in Screening shRNA Recombinant Clone

以单菌落为模板进行PCR扩增,直接筛选插有发夹RNA(Small hairpin RNA,shRNA)干扰片段的重组阳性克隆。以圆滑单菌落为PCR模板,采用载体通用引物直接扩增目的片段,质粒测序验证PCR结果的正确性。结果表明,在筛选的6个菌落中均扩增出324 bp的目的条带,进一步进行测序分析鉴定,证实了菌落PCR阳性克隆含有发夹RNA干扰片段。单菌落PCR是一种简便、快速、可靠、有效筛选重组阳性克隆的方法。

The individual bacterial colonies were adopted for templates of PCR to screen recombinant clone carrying small hairpin RNA. The PCR amplification with individual bacterial colonies and universal primer was developed to detect targeting fragment. The positive colonies were further confirmed by sequencing. Results showed that among the six clones, the positive band in the size of 324 bp were detected on agarose gel. DNA sequencing confirmed that shRNA template was cloned into plasmid vector successfully. Individual bacterial colony PCR is a simple,rapid,reliable and efficient method for screening the recombinant clones.