Macherey-Nagel NucleoSpin 740727.10 User Manual page 17

Suboptimal elution conditions
• If possible, use a slightly alkaline elution buffer like Buffer AE
Poor
plasmid
yield
(continued)
Low copy-number plasmid was used
• At least double or triple culture volume and increase lysis buffers
Reagents not applied properly
• Add indicated volume of 96–100 % ethanol to Buffer AQ
Nuclease-rich host strains used
• Especially when working with nuclease-rich strains, keep
No plasmid
yield
Inappropriate storage of plasmid DNA
• Quantitate DNA directly after preparation, for example, by
Nicked plasmid DNA
• Cell suspension was incubated with alkaline Lysis Buffer A2 too
Genomic DNA contamination
Poor
• Cell lysate was vortexed or mixed too vigorously after addition
plasmid
quality
Smeared plasmid bands on agarose gel
• Especially when working with nuclease-rich strains, keep
Plasmid DNA purification
(5 M Tris / H Cl, pH 8.5). If nuclease-free water is used, check the
pH of the water. Elution efficiencies drop drastically with buffers
< pH 7.
if final amount of cells exceed the recommended volumes of
table 3.
Concentrate and mix thoroughly (see section 3).
plasmid preparations on ice or frozen in order to avoid DNA
degradation.
agarose gel electrophoresis. Store plasmid DNA dissolved in
water at < -18 °C or at < +5 °C when dissolved in Buffer AE or
TE buffer.
long. Reduce lysis time.
of Buffer A2. Genomic DNA was sheared and thus liberated.
plasmid preparations on ice or frozen in order to avoid DNA
degradation.
MACHEREY-NAGEL – 08/ 2 013, Rev. 01 17