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Affinity purification of plasmid DNA directly from crude bacterial cell lysates
Author(s) -
Darby Richard A.J.,
Forde Gareth M.,
Slater Nigel K.H.,
Hine Anna V.
Publication year - 2007
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.21492
Subject(s) - plasmid , dna , plasmid preparation , lysis , genomic dna , recombinant dna , microbiology and biotechnology , dna supercoil , biology , chromatography , affinity chromatography , chemistry , biochemistry , gene , dna replication , enzyme , pbr322
Abstract We have shown previously that a sequence‐specific DNA‐binding protein based on the Lac repressor protein can isolate pre‐purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with impractically low DNA yields. We have optimized the procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100–150 µg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open‐circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re‐hydrated for use when required. Biotechnol. Bioeng. 2007;98: 1103–1108. © 2007 Wiley Periodicals, Inc.