Plasmid DNA Purification
MACHEREY-NAGEL – 01/2008/ Rev. 04
63
Problem
Possible cause and suggestions
NucleoBond
®
Xtra column
filter clogs
during filtra-
tion
Culture volumes are too large
•
Refer to section 4.4-4.6 regarding recommended culture vol-
umes and larger lysis buffer volumes.
Precipitate was not resuspended before loading
•
Invert crude lysate at least 3 times directly before loading.
Incomplete precipitation step
•
Make sure to mix well after neutralization to completely precipi-
tate SDS and chromosomal DNA.
NucleoBond
®
Xtra column is
blocked or
very slow
Sample is too viscous
•
Do NOT attempt to purify lysate prepared from a culture volume
larger than recommended for any given column size with stan-
dard lysis buffer volumes. Incomplete lysis not only blocks the
column but can also significantly reduce yields. Refer to section
4.4 and 4.5 for recommended culture volumes and section 4.6
for larger culture volumes and adjusted lysis buffer volumes.
•
Make sure to mix well after neutralization to completely precipi-
tate SDS and chromosomal DNA.
Lysate was not cleared completely
•
Use NucleoBond
®
Xtra column filter or centrifuge at higher speed
or for a longer period of time.
•
Precipitates occur during storage. Clear lysate again before
loading the column.
Genomic DNA
contamination
of plasmid
DNA
Lysis treatment was too harsh
•
Make sure not to lyse in Buffer LYS for more than 5 min.
Lysate was mixed too vigorously or vortexed after lysis
•
Invert tube for only 5 times. Do not vortex after addition of LYS.
•
Use larger tubes or reduce culture volumes for easier mixing.
