An automated process to extract plasmid DNA by alkaline lysis.
With advances in the development of DNA vaccines and gene therapy, there is a growing need for plasmid DNA with high quality for fundamental research and clinical trials. In this report, a scalable automated process for large-scale preparation of plasmid is described. This process is based on alkaline lysis and can be easily scaled up to meet demands for larger quantities. In the process, harvested bacteria are passed through two mixing chambers at controlled speeds to affect lysis and control alkalinity. The resulting solution is passed through a series of filters to remove contaminants, and ethanol precipitated. System parameters are examined to maximize the quantity and quality of the prepared plasmid. Using this procedure, plasmid can be extracted and purified from 1 l of Escherichia coli cultures at an OD600 nm of 50 in less than 45 min. The plasmid yields are approximately 90 mg/l culture.
Li
Xiaolin
X
State Key Laboratory for Agro-Biotechnology, College of Biological Science, China Agricultural University, Beijing, 100094, China.
Jin
Huali
H
Wu
Zhifang
Z
Rayner
Simon
S
Yin
Jiangmei
J
Yu
Yang
Y
Zhang
Wenjuan
W
He
Zhonghuai
Z
Wang
Chen
C
Wang
Bin
B
D001331:Automation / Q000295:instrumentation*; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D010957:Plasmids / Q000302:isolation & purification*; D012972:Sodium Hydroxide
D004269:DNA, Bacterial; D012972:Sodium Hydroxide