应用以菌落为模板的聚合酶链反应技术筛选重组阳性克隆

Screen for recombinant clones by colony Polymerase chain reaction

目的 应用以菌落为模板的聚合酶链反应 (PCR)技术筛选插有小鼠Doc 1R基因组序列的重组阳性克隆。方法 应用扩增小鼠Doc 1R基因组序列时使用的引物 ,以基因重组扩增后得到的菌落为模板 ,直接进行PCR扩增。结果 在筛选的 5个菌落中有 3个可见到 15 0 0bp大小的阳性条带与Doc 1R基因片段大小一致的阳性克隆 ,并对此 3个阳性克隆分别提取质粒 ,进一步进行双酶切及序列分析鉴定 ,证明结果正确。结论 菌落PCR用于筛选重组阳性克隆是一种简便、快速、可靠。

Objective To screen the Doc 1R gene recombinant plasmid by use of colony PCR. Method The recombinant colonies were transfered into the PCR reaction mixture. The PCR primers were used for constructing mouse Doc 1R genomic sequence. Result Among the 5, 3 positive strips in the size of 1 500 bp were visible, which were the same as the Doc 1R gene fractions in terms of their sizes were screened as positive clones. The positive colony were further confirmed by double digestion and DNA sequencing. Conclusion Colony PCR is a simple, efficient and reliable technique for screening the recombinant.