摘要
目的:建立简便易行、成本低廉和安全可靠的高纯度质粒大量制备方法,探讨其在分子生物学研究中的应用。方法:培养含目的质粒的菌株,运用改良后的碱裂解法大量提取质粒DNA并进行纯化,微量核酸仪测定质粒DNA的产量及纯度;采用琼脂糖凝胶电泳、限制性核酸内切酶酶切、转染哺乳动物细胞和慢病毒包装,鉴定质粒DNA的质量及转染效率。结果:本质粒提取方法获得质粒DNA产量为2.5 mg/L菌液,OD260/OD280=1.91;以超螺旋质粒DNA为主;限制性核酸内切酶可完全酶切;哺乳动物细胞转染效率在85%以上;可用于慢病毒包装。结论:本方法抽提质粒DNA操作简单、经济实用,可替代质粒大量提取试剂盒获得高产优质的质粒DNA,充分满足了分子生物学实验的要求。
Objective： To develop an efficient,economical and environmental method for the preparation of highly purified plasmid DNA in high yield, and explore its application value in molecular biological research. Methods： Plasmid DNA was isolated and purified by an improved general alkaline lysis method. The purified plasmid DNA was quantified by spectrophotometric measurement, and identified and evaluated by electrophoresis in agarose gel, digested with restriction endonuclease, transfected into mammalian cells and lentivirus packaged. Results： The quantity of plasmid DNA prepared by the optimized method was 2.5 mg per milliliter culture of transformed bacteria, and the ratio of OD260/OD280 was 1.91. The plasmid DNA was mainly composed of supercoiled structure. The plasmid DNA could be fully digested with restriction endonuclease. The transfection efficiency of the plasmid DNA in mammalian cells with Lipofectamine 2000 was beyond 85%. Conclusion： The improved method is simple, practical and economical. It can replace the ＂Plasmid Maxi Preparation Kits＂ method to prepare sufficient plasmid DNA to meet the needs of molecular biological resarches.
出处
《泸州医学院学报》
2014年第3期257-260,共4页
Journal of Luzhou Medical College
关键词
质粒DNA
制备
酶切
转染
慢病毒包装
Plasmid
Preparation
Restriction endonuclease digestion
Transfection
Lentivirus package
