关键词: 质粒提取, 质粒构型, 低温, 转染效率

Abstract: 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°C, and the extraction process was splitted 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 obtained to increase plasmid yield and working efficiency in mammalian cells.

Key words: plasmid extraction, plasmid isoform, low temperature, transfection efficiency