Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04
21
4.12
Determination of DNA yield and quality
The
yield
of a plasmid preparation should be estimated prior to and after the isopro-
panol precipitation in order to calculate the recovery after precipitation and to find the
best volume to dissolve the pellet in. Just use either
NucleoBond
®
Xtra
Elution
Buffer ELU or the respective low-salt buffer as a blank in your photometric measure-
ment.
The nucleic acid
concentration
of the sample can be calculated from its UV absor-
bance at 260 nm where an absorbance of 1 (1 cm path length) is equivalent to 50 µg
DNA/ml. Note that the absolute measured absorbance should lay between 0.1 and
0.7 to be in the linear part of Lambert-Beer´s law. Dilute your sample in the respec-
tive buffer if necessary.
The plasmid
purity
can be checked by UV spectroscopy as well. A ratio of A
260/280
between 1.80-1.90 and A
260/230
around 2.0 indicates pure plasmid DNA. An A
260/280
ra-
tio above 2.0 is a sign for too much RNA in your preparation, an A
260/280
ratio below
1.8 indicates protein contamination.
Plasmid
quality
can be checked by running the precipitated samples on a 1% aga-
rose gel. This will give information on conformation and structural integrity of isolated
plasmid DNA i.e. it shows whether the sample is predominantly in the favorable su-
per-coiled (ccc, usually the fastest band), an open circle (oc) or even linear form (see
section 8.1, Figure 6).
4.13
Convenient stopping points
Cell pellets can easily be stored for several months at -20°C.
Cleared lysates can be kept on ice or at 4°C for several days.
Once the column purification is started it should not be interrupted for more than an
hour. The columns can be left unattended for several minutes since they do not run
dry but complete drying out due to evaporation should be avoided at any rate. You
should proceed to the elution step after which the eluate can be stored for several
days at 4°C. Note that the eluate should be warmed up to room temperature before
precipitating the DNA to avoid co-precipitation of salt.
