| |
POSSIBLE CAUSE |
RECOMMENDATIONS |
| Transformation
efficiencies lower than expected |
There may be impurities present
in the DNA. |
Ensure that the DNA does not contain any protein,
detergents or ethanol. |
| There may be too much DNA. |
Transformation efficiencies are
calculated using 10pg pUC19. Transformation efficiencies will decrease with increasing
amounts of DNA; however, total numbers of colonies will increase with the amount
of DNA. |
| The cells may have been improperly
handled or stored. |
Cells should be thawed in ice and used immediately.
Do not refreeze cells and do not vigorously mix cells by vortexing. Cells must
be stored at -70°C to guarantee stability.
Transformation
should be done in the tube/well that contains the cells.
Transformation
should be done according to the recommended protocol to ensure the guaranteed
transformation efficiency. |
| You may be using a large plasmid. |
Transformation efficiencies are
calculated using pUC19. Larger plasmids will result in lower transformation efficiencies. |
 Top
of Page |
| |
POSSIBLE CAUSE |
RECOMMENDATIONS |
| Few
or no colonies seen after ligation |
You may have too little DNA in your
ligation mix. |
Increase the amount of vector and/or insert in
your ligation. |
| The ligation may have been inefficient. |
Double check the cloning strategy,
enzymes and concentrations of both vector and insert. Set up a control for each
step of the cloning procedure. |
| You may have the wrong antibiotic
concentration. |
Some plasmids with very low copy
number require lower antibiotic concentrations than standard high copy number
plasmids. Check the antibiotic concentration that is optimal for your vector. |
| You may have restriction enzymes or phosphatase
remaining in your ligation mix. |
Make sure that you inactivate or eliminate any
trace of restriction enzymes or phosphatase before ligating your DNA. |
| You may have diluted your transformation too
much. |
After ligation, calculated transformation efficiencies
are usually much lower than transformation efficiencies calculated with supercoiled
DNA. You may plate most of your transformations to ensure that you get colonies
and dilute the rest to ensure that you obtain single colonies. |
| Top
of Page |
| |
POSSIBLE CAUSE |
RECOMMENDATIONS |
| Problems with blue and white
screening |
You may have incorrect amounts of X-gal and/or IPTG added
to your agar plates. |
Agar plates should include the appropriate antibiotic, 40µg/ml
X-gal and 1mM IPTG. |
| Top
of Page |
| |
POSSIBLE CAUSE |
RECOMMENDATIONS |
| Satellite colonies |
The antibiotic concentration may be incorrect. |
Check the antibiotic concentration added to the plates. |
| |
The ampicillin may be degraded. |
Make sure that ampicillin was not added to the medium when
it was still too hot and that it was properly stored. Use carbanicillin, an antibiotic
similar to ampicillin with improved stability. |
| |
You may have too many colonies in the plate. |
Plate the transformation at a higher dilution. |
| |
You many have incubated the plates too long. |
Incubate the plates overnight at 37°C or for 2-3 days
at room temperature in the case of temperature-sensitive plasmids. |
| Top
of Page |
Trouble Shooting Guide
Diagrams and Protocols
Get
Adobe Acrobat Reader for PDFs