Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04
17
4.9
Filtration and loading of the lysate
After the alkaline lysis, the sample has to be cleared from cell debris and precipitate
to ensure high plasmid purity and a fast column flow rate. This is achieved by passing
the solution through a
NucleoBond
®
Xtra column filter
which is provided already
inserted into the
NucleoBond
®
Xtra column
.
Midi
Maxi
The
NucleoBond
®
Xtra column filters
are designed to eliminate the centrifugation
step after alkaline lysis. They are pre-wet during column equilibration and allow a
time-saving simultaneous clearing of bacterial lysate and loading of the
Nu-
cleoBond
®
Xtra column
.
Compared to lysate clearing by centrifugation or syringe filters the
NucleoBond
®
Xtra column filter
furthermore avoids shearing of large DNA constructs such as
PACs or BACs by the gentle depth filter effect (filtration occurs on the surface of the
filter as well as inside the filter matrix). Its special material and design lead to very
rapid passage of the lysate through the filter and even very large lysate volumes can
be applied without the risk of clogging. This is especially important for e.g. low-copy
plasmid purification. However, if more than the recommended cell mass (see section
4.4, Table 2, section 4.5, Table 3) was lysed, it might be advantageous to use a cen-
trifuge for lysate clarification rather than the provided column filters due to their lim-
ited precipitate capacity.
4.10
Washing of the column
The high salt concentration of the lysate prevents proteins and RNA from binding to
the
NucleoBond
®
Xtra column
(see section 4.2, Figure 2). However, to remove all
traces of contaminants and to purge the dead volume of the
NucleoBond
®
Xtra col-
umn filters
it is important to wash the column and the filter in two subsequent wash-
ing steps.
First apply Buffer EQU to the funnel rim of the filter to wash all residual lysate out of
the filter onto the column. Do not just pour the buffer inside the filter. Then pull out
and discard the column filter or remove the filter by turning the column upside down.
It is essential to wash the
NucleoBond
®
Xtra column
without filter for a second time
with Wash Buffer WASH. This ensures highest yields with best achievable purity.
NucleoBond
®
Xtra
column filter
NucleoBond
®
Xtra
column
