一种提高质粒转染效率的质粒提取方法的优化

Optimization of plasmid DNA extraction method for improvement of transfection efficiency

目的优化和改进质粒提取方法,以提高质粒的提取效率,增加其在哺乳动物细胞中的表达效率。方法分别在室温条件和4℃条件提取质粒,并将细菌裂解、过柱和洗脱3个过程分解在两种温度下提取质粒;使用紫外分光光度计和琼脂糖凝胶电泳分别检测质粒浓度和超螺旋形式;将质粒转染哺乳动物细胞,使用荧光显微镜和Western blot检测荧光蛋白的表达水平。结果低温进行质粒提取能够显著提高质粒提取效率(P<0.05),并显著提高其在哺乳动物细胞中的表达效率(P<0.01)。质粒提取的3个步骤:细菌裂解、过柱和洗脱均对温度敏感,从而影响质粒的得率和在哺乳动物细胞中的表达效率。结论成功获得一种简单易行的提高质粒得率和在哺乳动物细胞中表达效率的质粒提取方法。

Objective To optimize an extraction method for isolation of plasmid DNA to improve extraction and transfection efficiency of plasmid DNA. Methods The plasmid was extracted at room temperature and 4 ℃, and the extraction process was programed as to bacterial lysis, column and elution at two temperatures. Plasmid concentration and quality were detected by UV spectrophotometer and agarose gel electrophoresis,respectively. The working efficiency of plasmid was detected by fluorescence microscopy and Western blot after transfecting into mammalian cells. Results Plasmid extraction at low temperature significantly improved the plasmid yield and increased its working efficiency in mammalian cells. The three steps of plasmid extraction were sensitive to temperature, including bacterial lysis, absorption and elution, which all contributed to the yield and quality of extracted plasmid. Conclusions A simple and convenient plasmid extraction method is successfully developed to increase plasmid yield and working efficiency in mammalian cells.