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6
Appendix
6.1
Troubleshooting
Problem
Possible cause and suggestions
Cell pellet not properly resuspended
• It is essential that the cell pellet is completely resuspended
Incomplete
SDS in Buffer A2 precipitated
lysis of
• Storage of Buffer A2 below 20 °C may cause precipitation of
bacterial
cells
Too many bacterial cells used
• See table 3 for maximum amount of cells.
Incomplete lysis of bacterial cells
• See „Possible cause and suggestions" above.
No or insufficient amounts of antibiotic used during cultivation
• Cells carrying the plasmid of interest may become overgrown
Poor
plasmid
yield
Bacterial culture too old
• Do not incubate cultures for more than 16 h (LB) or 12 h
Incomplete neutralization
• Mix thoroughly after addition of Buffer A3 until LyseControl has
16
Plasmid DNA purification
prior to lysis. No cell clumps should be visible before addition
of Buffer A2.
SDS. If salt precipitate is observed, incubate buffer at 30–40 °C
for several minutes and mix well until all precipitate is redissolved
completely. Cool down to room temperature before use.
by cells without plasmid (see table 2), when inadequate levels
of the antibiotics are used. Add appropriate amounts of freshly
prepared stock solutions to all media; both solid and liquid.
(rich media) at 37 ° C under shaking to avoid starvation and
plasmid degradation.
turned colorless without any traces of blue.
MACHEREY-NAGEL – 08 / 2 013, Rev. 01
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