Troubleshooting Tips

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.