一种改良式质粒DNA提取方法的建立与应用

目的建立一种稳定、可靠的质粒DNA提取改良方法,探讨其在分子生物学实验研究中的应用价值。方法将含有目的质粒的菌株扩增后,运用改良后的碱裂解法进行质粒DNA小量提取,微量核酸仪测定提取质粒DNA的产量及纯度;采用琼脂糖凝胶电泳、内切酶酶切及质粒转染真核细胞鉴定提取质粒DNA的质量及转染效率。结果改良式质粒提取方法获得质粒DNA产量为3.2 mg/L菌液,A260/280=1.91;内切酶酶切完全;质粒DNA以超螺旋结构为主;真核细胞转染效率为20%左右。结论改良式质粒DNA抽提方法操作简单、实用,获得质粒DNA产量多、质量高,能达到分子生物学常规实验的要求。

An improved and economical method for isolation of plasmid DNA

Objective To explore a simple,improved and inexpensive method for isolation of plasmid DNA and its value in molecular biological laboratory research.Methods Plasmid DNA was isolated using general alkaline lysis method and improved method,respectively.The isolated plasmid DNA was quantified by spectrophotometric measurement,and evaluated by the electrophoresis of plasmid DNA in an agarose gel,the restriction enzyme digestion,and the transfection into eukaryotic cells.Results The quantity of plasmid DNA isolated by the improved method was 3.2μg per milliliter culture of transformed bacteria,and the ratio of A260/280 was 1.91.The plasmid DNA could be fully digested by restriction enzyme.The efficiency of the plasmid DNA transfection into eukaryotic cells by Lipofectamine2000 was about 20%.Conclusion The improved method is simple,practical and economical.Plasmid DNA isolated by this method has been shown to be suitable for restriction enzyme digestion and cell transfection.