|
|
POSSIBLE CAUSE |
RECOMMENDATIONS |
| Poor
DNA yield
|
Poor cell
lysis due to large amount of
bacterial cells. |
 Use less rich
media like 2xYT.
Use static growth culture conditions.
Decrease incubation time. |
Poor cell
lysis due to inactivation of
enzymes in Lysis Solution/Enzyme
Mix. |
Store
Enzyme Mix at -20°C.
Store
Lysis Solution/ Enzyme Mix at 4°C.
Combined solution is stable for one
month.
Prepare fresh Lysis Solution/Enzyme Mix. |
Failure
to resuspend bacterial
cells completely. |
Increase vortexing or mixing
time until pellet is
completely dispersed. |
| Plasmid
did not propagate. |
Ensure
appropriate antibiotic was included during
all stages of growth. |
| Cell pellet too tight. |
Decrease centrifugation
force or time. |
| Failure to pellet cells. |
Increase centrifugation
time. |
| Over-drying of DNA
pellet. |
Decrease
drying time.
Increase the incubation time for elution. |
| Ethanol used instead
of Isopropanol. |
Use Isopropanol as
suggested. |
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of Page |
|
|
POSSIBLE CAUSE |
RECOMMENDATIONS |
| Large
amount of RNA
and/or genomic DNA
contamination.
|
Processing
too large of a cell pellet. |
Use
less rich media like 2xYT or LB.
Use
static growth culture conditions.
Decrease
incubation time. |
Inactivation
of enzymes in Enzyme Mix
or in Lysis Solution/Enzyme Mix. |
Store
Enzyme Mix at -20ºC.
Store Lysis Solution/ Enzyme Mix at 4°C.
Combined solution is stable for one
month.
Prepare fresh Lysis Solution/Enzyme Mix. |
| Top
of Page |
|
|
POSSIBLE CAUSE |
RECOMMENDATIONS |
| Unfocused
band
visualized on agarose gel.
|
dH2O
used to elute DNA. |
Use 10mM Tris-HCl, pH
8.0 or TE0.1 instead of
dH2O to elute DNA. |
| |
|
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of Page |
|
|
POSSIBLE CAUSE |
RECOMMENDATIONS |
| Inflated
OD260 values.
|
Presence
of small RNA fragments. |
DNA
quantification via agarose gel
electrophoresis or Invitrogen Quant-iT™
PicoGreen® dsDNA Assay Kit.
Additionally, clean purified plasmid DNA with
Edge BioSystems QuickStep™2 PCR
Purification Kit. |
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of Page |
|
|
POSSIBLE CAUSE |
RECOMMENDATIONS |
| Degradation
seen in
restriction analysis.
|
Poor sample
prep. |
Follow recommended protocols
carefully. |
Incomplete
removal of lysate and
washes. |
Use inverted
spin to remove lysate and washes. |
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of Page |
|
|
POSSIBLE CAUSE |
RECOMMENDATIONS |
| Poor
sequencing results.
|
Poor sample
prep. |
Follow recommended protocols
carefully. |
| Too much
or too little DNA is used. |
Adjust
the DNA input based on the BigDye®
volume and total reaction volume. |
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of Page |
Diagrams
and Protocols
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