| |
POSSIBLE CAUSE |
RECOMMENDATIONS |
| Transformation
efficiencies lower than
expected
|
There may
be DNA impurities present. |
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 number of clones 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 -80°C to guarantee stability.
Transformation should be done according to
the recommended protocol to ensure the
guaranteed transformation efficiency. |
You may be using a
very
large plasmid. |
Transformation efficiencies are calculated
using
pUC19. Larger plasmids will result in lower
transformation efficiencies. |
| The ligation may have
been inefficient. |
Double check the cloning strategy, enzymes
and
concentrations of vector and insert. Set up a
control for each step of the cloning procedure. |
You may have too little
DNA in
your ligation mix. |
Increase the amount of vector and/or insert
in your
ligation. |
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 the wrong
antibiotic
concentration. |
Some plasmids with very low copy number
require
lower antibiotic concentrations than standard high
copy number plasmids. Verify the antibiotic
concentration is optimal for your vector. |
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. |
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| |
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. Check
that plates are not more than one month old or
have been stored above 4°C. |
You may
have too many
colonies in the plate. |
Plate the
transformation at a higher dilution. |
You may have incubated
the plates too long. |
Incubate the plates
overnight (16-18 hours) at 37°C
or for 2-3 days at room temperature in the case of
temperature-sensitive plasmids. |
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| |
POSSIBLE CAUSE |
RECOMMENDATIONS |
Little or no protein
expression detected
following induction |
The vector may have the wrong
promoter or there may be a cloning
design problem. |
Ensure that the promoter
is compatible with
the T7 RNA polymerase-based expression
systems.
Ensure that the gene of interest has an
initiation codon or it is in the correct frame
with the fusion tag and stop elements.
Sequence the construct to ensure that no
undesired changes have been introduced
during the cloning steps. |
You may not have added any or
enough IPTG to induce protein
expression. |
Check the induction requirements
for your vector. |
The protein does not express
well in E. coli. |
Use a different expression system. |
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| |
POSSIBLE CAUSE |
RECOMMENDATIONS |
| Insoluble protein |
Temperature, IPTG concentration,
fusion tags and denaturing conditions
can affect solubility. |
Lower the temperature during
induction.
Lower the IPTG concentration.
Use a different fusion tag.
Use denaturing conditions to solubilize the
protein. |
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Trouble Shooting Guide
Diagrams and Protocols
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