Protocol abstract
Nature Protocols 3, - 176 - 180 (2008)
Published online: 17 January 2008 | doi:10.1038/nprot.2007.526
Subject Categories: Genetic modification | Isolation, purification and separation | Nucleic acid based molecular biology
A continuous process to extract plasmid DNA based on alkaline lysis
Xiaolin Li1, Huali Jin1, Zhifang Wu1, Simon Rayner1,2 & Bin Wang1
Abstract
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 
80–90 mg l-
1 culture.
- State Key Laboratory for Agro-Biotechnology and the Key Laboratory of Agro-Microbial Resources and Applications of MOA, China Agricultural University, Beijing 100094, China.
- Present address: Wuhan Institute of Virology, Chinese Academy of Science, Wuhan, Hubei 430072, China.
Correspondence to: Bin Wang1 e-mail: bwang3@cau.edu.cn
nature-products
A-Z product listing
- 4-Channel peristaltic pump(Hitachi Koki)
- 70% Ethanol(Beijing Chemical Factory)
- Acetic acid(Beijing Chemical Reagents Company)
- Agarose(Biowest)
- DNA markers(TaKaRa)
- EDTA(Sigma)
