Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04
62
Problem
Possible cause and suggestions
No or low
plasmid DNA
yield
Plasmid did not propagate
•
Check plasmid content in the cleared lysate (see Figure 6). Use
colonies from fresh plates for inoculation and add selective anti-
biotic to plates and media.
•
Estimate plasmid content prior to large purifications by a quick
NucleoSpin
®
Plasmid or NucleoSpin
®
Plasmid QuickPure prepa-
ration.
Alkaline lysis was inefficient
•
Too much cell mass was used. Refer to section 4.4-4.6 regard-
ing recommended culture volumes and lysis buffer volumes.
Check plasmid content in the cleared lysate (see Figure 6).
•
Check Buffer LYS for SDS precipitation before use, especially
after storage below 20°C. If necessary incubate the bottle for
several minutes at 30-40°C and mix well until SDS is re-
dissolved.
SDS- or other precipitates are present in the sample
•
Load the crude lysate onto the NucleoBond
®
Xtra column filter
inserted in the NucleoBond
®
Xtra column. This ensures com-
plete removal of SDS precipitates. Incubation of cleared lysates
for longer periods of time might lead to formation of new pre-
cipitate. If precipitate is visible, it is recommended to filter or
centrifuge the lysate again directly before loading it onto the Nu-
cleoBond
®
Xtra column.
Sample/lysate is too viscous
•
Too much cell mass was used. Refer to section 4.4-4.6 regard-
ing recommended culture volumes and lysis buffer volumes.
•
Make sure to mix well after neutralization to completely precipi-
tate SDS and chromosomal DNA. Otherwise, filtration efficiency
and flow rate go down and SDS prevents DNA from binding to
the column.
pH or salt concentrations of buffers are too high
•
Check plasmid content in the wash fractions (see Figure 6).
Keep all buffers tightly closed. Check and adjust pH of Buffer
EQU (pH 6.5) and ELU (pH 9.0) with HCl or NaOH if necessary.
