Invitrogen Bac to Bac Expression System

Troubleshooting, continued
Isolating Bacmid DNA, continued
Problem Reason Solution
Poor yield
Used incorrect antibiotic
concentrations
Grow transformed DH10Bac cells in
LB medium containing 50 µg/ml
kanamycin, 7 µg/ml gentamicin, and
10 µg/ml tetracycline.
Bacmid DNA contains a
mixture of recombinant
bacmid and empty
bacmid
Picked a colony that was gray or
dark in the center
Analyze more white DH10Bac
transformants and choose one that
contains recombinant bacmid DNA
only.
Transfecting
Insect Cells
The table below lists some potential problems and possible solutions that may
help you troubleshoot insect cell transfection.
Problem Reason Solution
Low yield of virus Low transfection efficiency • Use Invitrogen’s Cellfectin ®
Reagent for transfection.
• Perform transfection in
unsupplemented Grace’s Medium;
make sure that no supplements,
FBS, or antibiotics are present
during transfection.
• Harvest viral supernatant when
signs of infection are visible (i.e.
>96 hours post-transfection).
Cells plated too sparsely Plate insect cells at the recommended
cell density.
Used too much or too little
Cellfectin ® or other lipid reagent
Time of incubation with
DNA:lipid complexes too short
or too long
Recombinant bacmid DNA is
degraded
Optimize the amount of Cellfectin ® or
other lipid reagent used.
Optimize the incubation time (e.g. 3 to
8 hours).
• Check the quality of your
recombinant DNA by agarose gel
electrophoresis prior to
transfection.
• Prepare bacmid DNA using
Invitrogen’s S.N.A.P. MidiPrep Kit
(Catalog no. K1910-01) or use the
procedure provided on page 51.
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