User guide

Troubleshooting, continued
Transfection
The table below lists some potential problems and possible solutions that may
help you troubleshoot your transfection experiments.
Observation Reason Solution
Very few or no stably-
transfected cells
obtained
Improperly cultured cells
• Exactly follow procedures as outlined in
Thawing and Subculturing Cells
(page 15).
• Thaw a new batch of early-passage cells.
• Do not add antibiotics during transfection.
Cells not passed 24 hours before
transfection
Approximately 24 hours before transfection,
pass cells at 3 × 10
5
cells/mL.
FreeStyle
™
Max Reagent
handled incorrectly
• Store at 4°C. Do not freeze.
• Mix gently by inversion. Do not vortex.
Used poor quality expression
construct plasmid DNA (i.e.,
plasmid DNA from a mini-prep)
Do not use miniprep plasmid DNA for
transfection. Prepare midiprep plasmid DNA
with low endotoxin contamination.
DNA contaminated Sterilize DNA using a 0.22 µm filter.
Used pcDNA
™
3.3 for single-
subunit protein expression
Only clones that are expressed from
pOptiVEC
™
construct can be selected using
HT-deficient CD OptiCHO
™
medium and
subsequently amplified using MTX. Reclone
your gene of interest in pOptiVEC
™
TOPO
®
TA and retransfect your cells.
Cells experienced too much
stress during selection with CD
OptiCHO
™
medium containing
500 μg/mL of Geneticin
®
Selective Antibiotic.
Perform two rounds of selection on your
transfected cells, one with CD OptiCHO
™
medium and one with CD OptiCHO
™
medium
and 500 µg/mL of Geneticin
®
Selective
Antibiotic.
Continued on next page
64