Transfection Problem |
Possible Cause |
Suggested Solution |
Low transfection
efficiency. |
Not using the optimal
transfection reagent. |
Select the right
transfection reagent likely to result in the highest transfection
efficiency for your cell type. Refer to transfection tips by clicking
"Support" tab on our website. Alternatively contact our tech support at
tech@signagen.com |
Low transfection
efficiency. |
DNA-transfection reagent
complexes not or poorly formed. |
Never use serum during
complex formation step! Serum-free DMEM are pretty good for complex
formation. While we recommend using serum containing medium for transfections, transfection complex must be formed in the absence of
serum.
Note: Never use Opti-MEM which disrupts transfection complex. |
Low transfection
efficiency. |
Inhibitors were present
during transfection complex formation. |
Serum-free is essential
for transfection complex formation. Avoid to use the following chemicals
during complex formation step: high concentration phosphate, chondroitin
sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans. |
Low transfection
efficiency. |
Cationic lipid was
frozen. |
Do not use cationic lipid
reagents (LipoD293™, LipoJet™ and PowerFect™, etc) that have been frozen.
Store at +4 °C. |
Low transfection
efficiency. |
Suboptimal cell density.
|
For DNA transfection or
DNA/siRNA co-transfection,
cell density should be around 70% confluent at time of transfection. For
siRNA transfection, cell density should be
~50% confluent at time of transfection. |
Low transfection
efficiency. |
Not using the optimal
transfection reagent concentration, DNA concentration, incubation time or
cell density. |
Optimize these parameters
per transfection reagent protocol. Refer to transfection tips by clicking
"Support" tab on our website. For further technical suggestions, please email
us at tech@signagen.com
|
Low transfection
efficiency. |
Problems with the
transfection assay. |
Include a positive
control for the transfection assay. |
Low transfection
efficiency. |
The promoter-enhancer of
the transfected DNA is not recognized by the cell type. |
Make sure the transfected
DNA is compatible with the target cell type. |
Low transfection
efficiency. |
For GenMute™, GenJet™,
PowerFect™ and PepMute™ siRNA reagents, fail to use specific buffer to
dilute these siRNA reagents and siRNA. |
For GenMute™, GenJet™,
PowerFect™ and PepMute™ siRNA reagents, be sure to use their pre-optimized
specific buffer to dilute these siRNA reagents and siRNA. |
Low transfection
efficiency. |
Keep transfection complex
at RT too long before adding to cells culture dish. |
The optimal complex
formation occurs from 10~20 minutes at RT for most of our DNA/siRNA
transfection reagents. Never leave the complex at RT longer than 30
minutes. |
Low transfection
efficiency. |
Cell density too low for
LipoD293™ reagent transfection. |
Use cells at ~70% confluency for LipoD293™ reagent transfections. |
Low transfection
efficiency. |
Too high spin speed
during making transfection complex. |
Protocols recommend
vortex mixing transfection reagent and DNA or siRNA followed brief spin to
bring mix drops to the bottom of the tube. But the spin speed can
NOT be too high. 100xg at 5 seconds is enough. Too high spin
speed will lead to sediment of transfection complex and very low
efficiency. |
Low transfection
efficiency with hard-to-transfect cells such as MDCK, MB2231, SaoS-2, etc. |
Use wrong transfection
protocol. |
For GenJet™,
LipoD293™ and PolyJet™ reagents, please try advanced protocol on
hard-to-transfect cells like MDCK, MDA_MB231, SaoS-2, etc for better
efficiency. |
High cell death
(toxicity). |
Too much DNA.
|
Perform a dose-response
curve to determine the optimal amount of DNA. Include a cationic lipid
reagent in the dose-response transfections, as DNA alone has a minimal
effect on cell growth. |
High cell death
(toxicity). |
Too much transfection
reagent. |
Perform a dose-response
curve to determine the optimal amount of transfection reagent. Include DNA
with the transfection reagent in the dose-response experiment, as the
transfection reagent alone has a minimal effect on cell growth. |
High cell death
(toxicity). |
Too low cell density. |
For DNA transfection,
cell density should be ~70% confluent at time of transfection. For
siRNA transfection or DNA/siRNA co-transfection, cell density should be
50% to 70% confluent at time of transfection. Low cell density may
see more significant cell death. |
High cell death
(toxicity). |
Cell viability decreased
or severe cell death. |
All our transfection
reagents are NOT interfered by serum and antibiotics, so perform
transfection in the presence of serum (10% FBS) and antibiotics. |
High cell death
(toxicity). |
For stable transfection,
selection antibiotic added too soon. |
Allow at least 48 h for
cells to express resistance before adding selective antibiotic.
|
High cell
death (toxicity) with PepMute™, GenMute™ or
PepMute™ Plus siRNA reagents. |
Use DMEM cell culture
medium supplemented with serum (FBS 10%) and antibiotics. |
Use DMEM/F12 or RPMI 1640
base medium supplemented with serum (FBS 10%) and antibiotics.
PepMute™, GenMute™ and PepMute™
Plus siRNA reagents are found to be toxic ONLY with DMEM base cell culture
medium.
|
Transfection not
reproducible. |
Confluency at the time of
transfection varied. |
Keep transfection
parameters such as confluency and phase of growth consistent. Transfection
not reproducible ,Cells changed in culture. If possible, thaw fresh
cells or obtain new cell line. |
Transfection reagent
solution is cloudy. |
Temperature is too low
during storage. |
Never freeze the
transfection reagent. Store at +4 °C. For GenJet™ series reagents, just
warm the reagents at 37 °C for several minutes until clear. It won't
compromise efficiency. |
Precipitation of
transfection complex. |
Excess EDTA present. |
Dissolve the DNA in water
rather than TE. |
Precipitation of
transfection complex. |
Excessive transfection
reagent and (or) DNA/siRNA during complex formation. |
Ensure concentrations of
transfection reagent and DNA/siRNA do not exceed recommended amounts in
complex formation. |
Poor silencing effect
during siRNA transfection |
Fail to use specific
buffer to dilute transfection reagent and siRNA |
For PepMute™,
GenMute™, GenJet™ and PowerFect™ siRNA transfection reagents, we recommend
using their specific transfection buffers to form transfection complex for
maximum silencing. Failure to use the buffers will lead to poor
silencing. |
Poor silencing effect
during siRNA transfection |
Fail to use optimal siRNA
concentration |
For PepMute™,
GenMute™, GenJet™ and PowerFect™ siRNA transfection reagents, we recommend
using final 1.0~100 nM siRNA for optimal silencing. For most
adherent mammalian cells, start from 5.0 nM of siRNA (which usually gives
satisfied silencing) while for hard-to-transfect cells, start from 50 nM
siRNA. |
Poor silencing effect
during DNA/siRNA co-transfection |
Fail to use specific
buffer to dilute siRNA transfection reagent, DNA and siRNA. |
For PepMute™,
GenMute™, GenJet™ and PowerFect™ siRNA transfection reagents, we recommend
using their specific transfection buffers to form transfection complex for
maximum silencing. |
Poor silencing effect
during DNA/siRNA co-transfection |
siRNA concentration is
too high or too low. |
For PepMute™,
GenMute™, GenJet™ and PowerFect™ siRNA transfection reagents, we recommend
using 1.0 ~ 20 nM of siRNA for DNA/siRNA co0transfection. Too high
siRNA concentration will produce flooding effect while too low siRNA
concentration will be NOT sufficient enough to generate good silencing. |