Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04
18
4.11
Elution and concentration of plasmid DNA
Elution is carried out under high-salt conditions and by a shift of pH from 6.5 to 9.0.
Under these alkaline conditions the positive charge of the anion-exchange resin is
neutralized and plasmid DNA is released. For any downstream applications it is nec-
essary to precipitate the DNA and to remove salt and all traces of alcohol since they
disturb or inhibit enzymatic activity needed for restriction or sequencing reactions.
All
NucleoBond
®
Xtra
eluates already contain enough salt for an isopropanol pre-
cipitation of DNA. Therefore the precipitation is started by directly adding 0.7 volumes
of isopropanol. To prevent co-precipitation of salt, use
room
-
temperature
(20-25°C)
isopropanol
only and do not let the plasmid DNA solution drop into a vial with iso-
propanol but
add isopropanol to the final eluate and mix immediately
.
Afterwards either follow the centrifugation protocol given after the
NucleoBond
®
Xtra
purification protocol or follow the support protocol for the
NucleoBond
®
Finalizers
in
section 7.3 to eliminate the time-consuming centrifugation steps for precipitation (use
of
NucleoBond
®
Finalizers
is only recommended for vector sizes smaller than
50 kbp).
The
NucleoBond
®
Finalizers
are designed for quick concentration and desalination
of plasmid and cosmid DNA eluates that are obtained by anion-exchange chromatog-
raphy based DNA purification. The sample is precipitated with isopropanol as men-
tioned above and loaded onto a special silica membrane by means of a syringe. After
an ethanolic washing step the membrane is dried by pressing air through the filter.
Elution of pure DNA is carried out with slightly alkaline low salt buffers like
Buffer
TRIS
(5 mM Tris/HCl, pH 8.5, provided with all
NucleoBond
®
Xtra Plus
kits) or TE
buffer (10 mM Tris/HCl, pH 7.5, 1 mM EDTA).
For maximum yield it is recommended to perform the elution step twice.
The
first elution step is done using fresh buffer whereas in the second elution step the
eluate from the first elution is reapplied on the
NucleoBond
®
Finalizer
to allow com-
plete solubilization of the plasmid.
DNA recovery highly depends on the used elution buffer volume.
Large volumes
result in a high recovery of up to 90% but in a lower DNA concentration. Small elution
volumes on the other hand increase the concentration but at the cost of DNA yield.
If a small volume is chosen, make sure to recover as much eluate as possible from
the syringe and
NucleoBond
®
Finalizer
by pressing air through the
NucleoBond
®
Finalizer
several times after elution and collecting every single droplet to minimize
the dead volume.
Figure 4 and 5 illustrate exemplarily how DNA recovery and final DNA concentration
depend on the buffer volume which is used for elution of DNA from the
NucleoBond
®
Finalizer
and
NucleoBond
®
Finalizer Large,
respectively
.
