Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04
13
The
type of plasmid
, especially the
size and the origin of replication (ori)
has a
crucial influence on DNA yield. In general, the larger the plasmid or the cloned insert
is, the lower is the expected DNA yield due to a lower copy number. Even a high-
copy construct based on a ColE1 ori can behave like a low-copy vector in case of a
large or unfavorable insert. In addition, the ori itself influences the yield by factor
10-100. Thus plasmids based on e.g. pBR322 or pACYC, cosmids or BACs are
maintained at copy numbers < 20 down to even only 1, whereas vectors based on
e.g. pUC, pBluescript or pGEM can be present in several hundred copies per cell.
Therefore, all the above mentioned factors should be taken into consideration if a
particular DNA yield has to be achieved. Culture volume and lysis procedures have to
be adjusted accordingly.
4.4
Culture volume for high-copy plasmids
Due to the influence of growth media (TB, CircleGrow, 2xYT), growth conditions
(shaking, temperature), host strain or type of plasmid insert etc. the final amount of
cells in a bacterial culture can vary over a wide range. By rule of thumb, 1 liter of
E.
coli
culture with an OD
600
of 1 consists of 1 x 10
12
cells and yields about 1.5-1.8 g cell
wet weight. Overnight cultures grown in LB medium usually reach an OD
600
of 3-6
under vigorous shaking in flasks. Fermentation cultures even reach an OD
600
of 10
and more. The expected DNA yield for a high-copy plasmid is approximately 1 mg
per gram cell wet weight.
It is therefore important to adjust the cell mass rather than the culture volume for the
best plasmid purification results. But since the cell mass or cell wet weight is tedious
to determine it was replaced in this manual by the mathematical product of optical
density at 600 nm (OD) and culture volume (V) - two variables that are much easier
to measure.
ODV = OD
600
x Vol [ml]
Note that the culture samples should be diluted if OD
600
exceeds 0.5 in order to in-
crease proportionally with cell mass. For a well grown
E. coli
culture a 1:10 dilution
with fresh culture medium is recommended. The measured OD
600
is then multiplied
with the dilution factor 10 to result in a theoretical OD
600
value. This OD
600
is used in
Table 2 to determine the appropriate culture volume.
Table 2: Recommended culture volumes for high-copy plasmids
Recommended culture volume for
NucleoBond
®
Xtra kit
Pellet
wet
weight
Rec.
ODV
OD
600
=
2
OD
600
=
4
OD
600
=
6
OD
600
=
8
OD
600
=
10
Midi
0.75 g
400
200 ml
100 ml
66 ml
50 ml
40 ml
Maxi
2.25 g
1200
600 ml
300 ml
200 ml
150 ml
120 ml
Table 2 shows recommended ODVs and the corresponding pairs of OD
600
and cul-
ture volume that can be easily handled using the standard kit protocol lysis buffer
volumes.
