Li Xiaolin, Jin Huali, Wu Zhifang, Rayner Simon, Wang Bin
State Key Laboratory for Agro-Biotechnology and the Key Laboratory of Agro-Microbial Resources and Applications of MOA, China Agricultural University, Beijing 100094, China.
Nat Protoc. 2008;3(2):176-80. doi: 10.1038/nprot.2007.526.
Rapid advances in the fields of DNA vaccines and gene therapy have produced increased demands for large quantities of recombinant plasmid DNA. The protocol presented here extracts plasmid DNA in a scalable continuous process based on an alkaline lysis protocol. In the process, harvested bacteria are passed through two mixing chambers at controlled speeds to effect lysis and control alkalinity. The resulting solution is passed through a series of filters to remove contaminants and then ethanol precipitated. This process replaces all the centrifugation steps before obtaining crude plasmid and can be easily scaled up to meet demands for larger quantities. Using this procedure, plasmid can be extracted and purified from 4 l of Escherichia coli culture at an OD 600 nm of 50 in <90 min. The plasmid yields are approximately 80-90 mg l(-1) culture.
DNA疫苗和基因治疗领域的快速发展使得对大量重组质粒DNA的需求不断增加。本文介绍的方案基于碱裂解法，以可扩展的连续过程提取质粒DNA。在此过程中，收获的细菌以可控速度通过两个混合室以实现裂解并控制碱度。所得溶液通过一系列过滤器以去除污染物，然后进行乙醇沉淀。此过程取代了获得粗质粒之前的所有离心步骤，并且可以轻松扩大规模以满足对更大数量的需求。使用此程序，可在不到90分钟的时间内从4升OD 600 nm为50的大肠杆菌培养物中提取和纯化质粒。质粒产量约为80 - 90 mg l(-1)培养物。
