| |
POSSIBLE CAUSE |
RECOMMENDATIONS |
|
Low
transformation efficiencies
|
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 numbers of clones will increase with
the amount of DNA. |
| The cells
may have been improperly handled or stored. |
Cells should be thawed on 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. |
| 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. |
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| |
POSSIBLE CAUSE |
RECOMMENDATIONS |
|
Little
or no protein expression detected following induction
|
The vector
may have a weak promoter or there may be a lack of good
cloning design. |
Ensure that the gene
of interest 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. |
| The concentration
of IPTG may be too low. |
Increase
the concentration of IPTG. |
| The protein
may be insoluble. |
Use denaturing
conditions to solubilize the protein. |
| The protein does
not express well in E. coli. |
Use a different expression
system. |
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| |
POSSIBLE CAUSE |
RECOMMENDATIONS |
| Cell
death |
The protein you are
trying to express may be toxic. |
You need tighter control over basal
expression; select a different expression host, like
BL21 (DE3) pLysE chemically competent cells.
|
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| |
POSSIBLE CAUSE |
RECOMMENDATIONS |
| Insoluble
protein |
Temperature, IPTG
concentration and fusion tags can affect solubility. |
Lower the temperature during induction
Lower the IPTG concentration.
Use a different fusion tag.
|
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| |
POSSIBLE CAUSE |
RECOMMENDATIONS |
| No
or very few colonies seen |
The protein
may be toxic and there may be high basal expression. |
You need
tighter control over basal expression and should select
a different expression host, like BL21 (DE3) pLysE chemically
competent cells. |
| You may
have the wrong antibiotic selection. |
Ensure that
the correct antibiotic selection was used. |
| The cells
may have lost competency. |
Test the
efficiency of the cells with the control DNA. |
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