重组大肠杆菌碱裂解方法的改进

Modification of Alkaline Lysis Method for Preparation of Plasmid DNAs from Recombinant E.coli

为了降低质粒DNA的生产成本,对经典碱裂解法中的溶液III进行了改进,以表达溶菌酶基因的pcDNKLYZ重组质粒转化的大肠杆菌DH5α为指示菌,用标准碱裂解和改进碱裂解法提取质粒pcDNKLYZ,以提取的质粒产量和质量为指标,判断优化碱性裂解法的性价比,结果显示,用改进后的碱裂解法裂解重组菌,提取的pcDNKLYZ质粒产量和质量等指标与标准方法接近,而成本仅为标准方法的1/4,可用于重组质粒的大规模制备。

In order to decrease the cost of the plasmid production, the standard alkaline lysis method for preparation of plasmid DNAs was modified, including the use of optimized volume of solution Ⅰ to resuspend the centrifuged bacteria and a new solution Ⅲ which contain less KAc to neutralize the cell lysate. Purification and quantitative analysis by BCA detection, Real-time PCR and agrose gel electrophoresis showed that both yield and quality of plasmid DNA prepared by the modified alkaline lysis remain unchanged compared to that by standard alkaline lysis method, but the cost was decreased to about 1/4 of normal method, which laid a solid foundation for large-scale production of recombinant plasmid DNAs for animal use.