Handbook of Size Exclusion Chromatography and Related ...

plasmid pBR322 and its phenol extract were separated on a TSKgel G6000PW
two-column system (each column 60 cm 7.5 mm ID). Plasmid pBR322 was
eluted between 27 and 31 minutes and was perfectly separated from RNA and
proteins, which were eluted after 36 minutes. Chromosomal DNA was also
removed fairly well, but not completely, because it was eluted continuously after
22 minutes. The purities of plasmid fractions collected from cleared lysate and
phenol extract were almost equivalent. The phenol extract sample was treated with
ATP-dependent deoxyribonuclease to digest linear double-stranded DNA-like
chromosomal DNA and was subjected to SEC on a TSKgel G6000PW column
(30 cm 7.5 mm ID). The result is shown in Fig. 7. The chromatogram suggests
that chromosomal DNA was almost completely eliminated from the plasmid
fraction. According to a purity test by agarose gel electrophoresis, the collected
plasmid fraction was free of RNA, proteins, and chromosomal DNA. The
separation between plasmid and other components was sufficient even when a
0.5 mL solution of the enzyme-treated phenol extract was applied to a column of
30 cm 7.5 mm ID and the separation was completed in about 15 minutes.
The major contaminants in the plasmid fraction obtained by SEC are
generally high molecular weight species such as E. coli chromosomal DNA and
rRNAs. The careful preparation of cell lysate would reduce the level of these
contaminants and, as a result, make it easy to separate these contaminants from
plasmids by SEC. The yields of plasmids would also be affected by the preparation
step of cell lysate (22).
Figure 7 Separation of phenol extract of cleared lysate of E. coli cells before (A) and
after (B) treatment with ATP-dependent deoxyribonuclease on a TSKgel G6000PW column
(30 cm 7.5 mm ID) in 0.1 M Tris–HCl buffer (pH 7.5) containing 0.3 M sodium chloride
and 1 mM EDTA at a flow rate of 1 mL/min. (From Ref. 21.)
© 2004 by Marcel Dekker, Inc.