Invitrogen Bac to Bac Expression System

Troubleshooting, continued
Cloning into the pFastBac Vectors, continued
Problem Reason Solution
No or few colonies
obtained after
transformation,
continued
Too much DNA transformed • For chemically competent cells,
add 1 to 10 ng of DNA in a volume
of 5 µl or less per 100 µl of cells. For
electrocompetent cells, add 10 to
50 ng of DNA in a volume of 1 µl
or less per 20 µl of cells.
• If you have purchased competent
cells, follow the manufacturer’s
instructions.
Incomplete ligation reaction • Optimize the ligation reaction.
• Include a ligation control (i.e.
digested pFastBac vector + ligase;
no insert). Check the ligation
reaction on a gel.
Note: Ligated products and linear
DNA transform 10X and 100-100X
less efficiently, respectively than
super-coiled DNA (Hanahan,
1983).
Ligation reaction mix inhibits
transformation of competent cells
Reduce the amount of ligation reaction
transformed. Dilute ligation reaction 5X
with TE Buffer prior to transformation.
Problem with antibiotic • Confirm use of the correct
antibiotic; confirm antibiotic
concentration.
• Check that the antibiotic is not
degraded (i.e. change in color of
solution or the appearance of
precipitate). Use fresh antibiotic.
Competent cells stored
improperly
Competent cells handled
improperly
Cells not heat-shocked or
incubated properly during
transformation
Store competent cells at -80°C.
Thaw cells on ice; use immediately
after thawing; do not vortex.
Follow the recommended
transformation procedure for the cells
you are using.
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