A continuous process to extract plasmid DNA based on alkaline lysis.
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.
Li
Xiaolin
X
State Key Laboratory for Agro-Biotechnology and the Key Laboratory of Agro-Microbial Resources and Applications of MOA, China Agricultural University, Beijing 100094, China.
Jin
Huali
H
Wu
Zhifang
Z
Rayner
Simon
S
Wang
Bin
B
D004269:DNA, Bacterial / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D005374:Filtration / Q000379:methods; D007218:Industrial Microbiology / Q000379:methods*; D010957:Plasmids / Q000302:isolation & purification*; D012972:Sodium Hydroxide
D004269:DNA, Bacterial; D012972:Sodium Hydroxide