User guide

Troubleshooting, continued
Protein Expression
The table below lists some potential problems and possible solutions that may
help you troubleshoot your protein expression levels.
Observation Reason Solution
No or low protein
detected in the
supernatant after
transient or stable
transfection
PCR primer does not contain
Kozak translation initiation
sequence
Add a Kozak consensus site to the forward
PCR primer, resynthesize your DNA and re-
clone. See the appropriate manual for each
TOPO
®
TA Cloning Kit for details.
Premature stop codons Remove stop codons by your method of
choice.
Improper or ineffective secretion
signal
Replace secretion signal. Use endogenous
secretion signal if possible.
Codons not optimized for
mammalian cells
Optimize codons for CHO-S cells.
Clone Selection
The table below lists some potential problems and possible solutions that may
help you troubleshoot your clone selection experiments.
Observation Reason Solution
Very few or no single
cell clones obtained
after cloning
Error in cell counting and
dilution during limiting dilution
cloning
• Follow procedures exactly as outlined in
Cell Counting and Dilution (pages 39 and
59).
• Dilute cells no more than 1:3 with a very
small amount (< 1mL) of cell suspension.
Conditioned Medium and HT
Supplements omitted from the
cloning medium
High cloning efficiency in LDC with CD
FortiCHO
™
Medium can be achieved with the
addition of conditioned media and HT
Supplement.
Conditioned Medium not
generated correctly
Follow procedures exactly as outlined in
Conditioned Medium Preparation, including
the cell seeding density and harvest day.
Plates moved too soon after
seeding the cells
Seeded plates should be incubated
undisturbed for 10–14 days.
Colonies not growing in CD
FortiCHO
™
Medium
Test CD OptiCHO
™
as the cloning medium.
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