三种无缝克隆试剂盒连接效率的比较

A comparison of the connection efficiency of three kinds of seamless cloning kits

目的比较生工生物工程(上海)股份有限公司、厂家A和厂家B三种无缝克隆试剂盒连接效率,为文库构建选择试剂盒提供参考。方法本研究采用Nco I和Xho I酶切pET26b获得线性化载体,PCR扩增制备线性化片段。无缝隙克隆反应体系转化感受态大肠杆菌,菌落PCR鉴定三种重组试剂盒连接阳性率。结果生工生物工程(上海)股份有限公司、厂家A和厂家B三种无缝克隆试剂盒分别长出107、86和16个克隆,阳性率分别为90.91%、72.73%和43.75%。结论从试剂盒性价比,转化总克隆数量和阳性克隆率等方面考虑,生工生物工程(上海)股份有限公司的无缝克隆试剂盒为文库构建的最佳选择。

Objective To compare the connection efficiency of three kinds of seamless cloning kits respectively of Sangon Biotech(Shanghai)Co.,Ltd.,Manufacturer A and Manufacturer B,so as to provide a reference for library construction and selection of kits.Methods In this study,pET26b went through restriction enzyme digestion by Nco I and Xho I to yield linearized vectors,and PCR amplification was used to prepare linearized fragments.The seamless cloning reaction system was used to transform competent Escherichia coli,and colony PCR was adopted to identify the connection positive rate of three recombinant kits.Results 107,86 and 16 clones were respectively obtained from the three kinds of seamless cloning kits of Sangon Biotech(Shanghai)Co.,Ltd.,Manufacturer A and Manufacturer B,with the positive rate of 90.91%,72.73%and 43.75%respectively.Conclusion In terms of cost performance of the kit,the total number of transformed clones and positive clone rate,the seamless cloning kits of Sangon Biotech(Shanghai)Co.,Ltd.are the best choices for library construction.