Invitrogen Bac to Bac Expression System

Troubleshooting, continued
Generating Recombinant Bacmid DNA, continued
Problem Reason Solution
Few colonies obtained Used LB medium for
Use S.O.C. Medium for the 4 hours
recovery/expression period growth time.
Poor blue/white colony
differentiation
Recovery/expression time too
short
Increase the recovery time to > 4 hours
at 37°C or 6 hours at 30°C.
Agar not at correct pH Adjust pH of LB agar to 7.0.
Intensity of the blue color too
weak
Too many or too few colonies on
plate
Incubation period too short or
temperature too low
IPTG concentration not optimal
• Use Bluo-gal, not X-gal.
• Increase the concentration of Bluogal
to 300 µg/ml.
• Use dark and light backgrounds to
view plates.
Adjust the serial dilutions of cells to
obtain an optimal number of colonies.
• Do not pick colonies until 48 hours
after plating.
• Incubate plates at 37°C.
Optimize the IPTG concentration. A
range of 20-60 µg/ml IPTG generally
gives optimal color development.
Isolating Bacmid
DNA
The table below lists some potential problems and possible solutions to help you
troubleshoot recombinant bacmid DNA isolation.
Bacmid DNA is
degraded
Problem Reason Solution
DNA stored improperly • Store purified bacmid DNA in
aliquots at –20°C.
• Do not freeze/thaw repeatedly.
High molecular weight bacmid
DNA handled improperly
• When isolating bacmid DNA, do
not vortex the DNA solution.
• Do not resuspend DNA pellets
mechanically; allow the solution to
sit in the tube with occasional
gentle tapping of the bottom of the
tube.
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