Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04
12
4.3
Growth of bacterial cultures
Yield and quality of plasmid DNA highly depend on the
type of culture media
and
antibiotics, the bacterial host strain, the plasmid type, size, and copy number.
For standard high-copy plasmids LB (Luria-Bertani) medium is recommended. The
cell culture should be incubated at 37°C with constant shaking (200-250 rpm) pref-
erably 12-16 h over night. Use flasks of at least three or four times the volume of the
culture volume to provide a growth medium saturated with oxygen. Alternatively, rich
media like 2xYT (Yeast/Tryptone), TB (Terrific Broth) or CircleGrow can be used. In
this case bacteria grow faster, reach the stationary phase much earlier than in LB
medium (
12 h), and higher cell masses can be reached. However, this does not
necessarily yield more plasmid DNA. Overgrowing a culture might lead to a higher
percentage of dead or starving cells and the resulting plasmid DNA might be partially
degraded or contaminated with chromosomal DNA. To find the optimal culture condi-
tions, the culture medium and incubation times have to be optimized for each host
strain/plasmid construct combination individually.
Cell cultures should be grown under
antibiotic selection
at all times to ensure plas-
mid propagation. Without this selective pressure, cells tend to lose a plasmid during
cell division. Since bacteria grow much faster without the burden of a high-copy
plasmid, they take over the culture rapidly and the plasmid yield goes down regard-
less of the cell mass. Table 1 gives information on concentrations of commonly used
antibiotics.
Table 1: Information about antibiotics according to Maniatis
Antibiotic
Stock solution
(concentration)
Storage
Working
concentration
Ampicillin
50 mg/ml in H
2
O
-20°C
50-100 µg/ml
Chloramphenicol
34 mg/ml in EtOH
-20°C
25-170 µg/ml
Kanamycin
10 mg/ml in H
2
O
-20°C
10-50 µg/ml
Streptomycin
10 mg/ml in H
2
O
-20°C
10-50 µg/ml
Tetracycline
5 mg/ml in EtOH
-20°C
10-50 µg/ml
The
E. coli
host strain
mostly influences the quality of the plasmid DNA. Whereas
strains like DH5
or XL1-Blue usually produce high quality super-coiled plasmid
DNA, other strains like e.g. HB101 with high levels of endonuclease activity might
yield lower quality plasmid giving poor results in downstream applications like enzy-
matic restriction or sequencing.
Maniatis T, Fritsch EF, Sambrook J:
Molecular cloning. A laboratory manual,
Cold Spring Harbor,
Cold Spring, New York 1982.
