一种改良式质粒DNA提取方法建立及应用.docVIP

一种改良式质粒DNA提取方法建立及应用 　 作者：张林琳，李磊，李翔，贺大林，朱国栋，王新阳 【关键词】 质粒DNA；提取；酶切；转染 　　摘要：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3.2mg/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。 　　关键词：质粒DNA；提取；酶切；转染 　　An improved and economical method for isolation of plasmid DNA 　　ABSTRACT: 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 3.2μg 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. 　　KEY WORDS: plasmid DNA; isolation; restriction enzyme digestion; transfection 　　质粒DNA是现代生物学实验中常用的载体工具，在基因工程领域应用非常广泛。质粒DNA的提取已成为分子生物学实验中的一种常规方法。在“碱裂解法”[1]的基础上进行优化改进，形成了一系列的质粒DNA提取方法[2]，并生产出一系列商品化提取试剂盒[3]。用经典“碱裂解法”少量提取质粒能获得高质量的质粒DNA，但在提取过程中，碱裂解不完全或裂解过度、蛋白质污染以及酚氯仿的残留都能影响进一步的分子生物学实验效果。我们研究所在“碱裂解法”的基础上进行了优化、改进，尝试了一种经济实用的改良式质粒DNA提取方法，经反复实践验证，该方法比较稳定、可靠，能获得转染级别的质粒DNA。 　　1 材料与方法 　　1.1 材料 　　1.1.1 质粒、菌株及培养液 含有绿色荧光蛋白报告基因(enhanced green fluorescence protein, EGFP)真核表达质粒载体pEGFPN1(质粒DNA全长4.9kb)购自Clontech公司；转化菌Ecoli.DH5a由本研究所保存。LB培养液(蛋白胨、酵母提取物、氯化钠)。