Affinity purification of plasmid DNA directly from crude bacterial cell lysates.

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 microg 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.

journal_name

Biotechnol Bioeng

authors

Darby RA,Forde GM,Slater NK,Hine AV

doi

10.1002/bit.21492

subject

Has Abstract

pub_date

2007-12-01 00:00:00

pages

1103-8

issue

5

eissn

0006-3592

issn

1097-0290

journal_volume

98

pub_type

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