Low transfection efficiency

Possible cause(s) Suggested solutions
Plasmid DNA, siRNA, or transfection reagent diluted in media containing serum or complexes formed in the presense of serumUse serum-free medium for dillutions of plasmid DNA, siRNA, and transfection reagents.

Note: we recommend using Opti-MEM® | Reduced Serum Medium (Cat. No. 31985-062) to dilute Lipofectamine® 2000 and DNA before complexing.
DNA: transfection reagent ratio
sub-optimal for cell line		Prepare complexes using a DNA (μg) to Lipofectamine® 2000 (μl) ratio of 1:2 to 1:3 for most cell lines. Optimization may be necessary. If so, vary DNA (μg): Lipofectamine® 2000 (μl) ratios from 1:0.5 to 1:5. If using a different transfection reagent, please consult the product manual.
Dilution times or complexing incubation period was too short
For Lipofectamine® 2000, we recommend that the DNA and transfection reagent be diluted separately and incubated for five minutes. Add the diluted reagents together and incubate for 20 minutes for optimal performance.
Not enough plasmid DNA used for dilution or complex formationVerify concentration using a second method or check the DNA for degradation. Determine DNA concentration by performing A260/A280 readings on a spectrophotometer or by using the Quant-iT™ DNA Assays Kits (Q33130, Q33120).
Plasmid DNA or siRNA used in transfection has degraded or is of poor quality
Ensure that the plasmid DNA or siRNA used for transfection is of high quality. For plasmid DNA purification kits, we recommend using our PureLink™ HiPure Nucleic Acid Purification Kits.
Cell density was not optimalLipofectamine® 2000 works best in cultures that are >90% at the time of transfection. When working with lower cell densities (<70%), we recommend Optifect™ Transfection Reagent as an alternative.
Complexes were added to cells in
serum-free medium		Try using growth medium containing serum when performing transfections. Transfection performance is typically better when the cells are more viable. If you require serum-free conditions, test medium for compatibility with Lipofectamine® 2000 since some serum-free formulations (e.g. CD293, SFM II, VP-SFM) may inhibit cationic lipid-mediated transfection.
Inhibitors were present in mediumDo not use antibiotics, EDTA, citrate, phosphate, RPMI, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the growth medium or in the medium used to prepare DNA:transfection reagent complexes.
Problems with assay used to measure efficiency or expressionUse a reporter gene to measure transfection efficiency. A reporter gene control allows you to confirm expression.
Promoter-enhancer on vector is not recognized by the cell typeVerify that the promoter-enhancer on your vector construct is compatible with the target cell type.
Cells have changed over time, or splitting conditions have changedIf transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
Transfection reagent stored improperlyWe recommend storing transfection reagents at 4°C. Freezing of transfection reagents, or storing them at room temperature, may decrease activity.

Reduced cell viability following transfection

Possible cause(s)
Suggested solutions
DNA: transfection reagent ratio
sub-optimal for cell line		Prepare complexes using a DNA (μg) to Lipofectamine® 2000 (μl) ratio of 1:2to 1:3 for most cell lines. Optimization may be necessary. If so, vary DNA (μg): Lipofectamine® 2000 (μl) ratios from 1:0.5 to 1:5.
Plasmid DNA preparation contains high levels of endotoxinEnsure that the plasmid DNA or siRNA used for transfection is of high quality. For plasmid DNA purification kits, we recommend using our PureLink™ HiPureNucleic Acid Purification Kits.
Cell density was not optimal
Lipofectamine® 2000 works best in cultures that are >90% at the time of transfection. When working with lower cell densities (<70%), we recommend Optifect™ Transfection Reagent as an alternative if cell viability is more important than expression levels.
Complexes were added to cells in
serum-free medium		Try using growth medium containing serum when performing transfections. Transfection performance is typically better when the cells are more viable. If you require serum-free conditions, test medium for compatibility with Lipofectamine® 2000 since some serum-free formulations (e.g. CD293, SFM II, VP-SFM) may inhibit cationic lipid-mediated transfection.
Complexes not thoroughly mixed in growth medium
Following addition of transfection complexes into medium, ensure that the plate or wells are thoroughly mixed to prevent concentration of DNA:transfection reagent complexes in the wells
Cells have changed over time, or splitting conditions have changedIf transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
Antibacterial agents were used in growth medium during transfectionDo not use antibiotics such as chloroquine, penicillin, or streptomycin in growth medium because during transfection, cells are more permeable to antibiotics, which may cause toxicity.
Transfection reagent stored improperlyWe recommend storing transfection reagents at 4°C. Freezing of transfection reagents, or storing them at room temperature, may decrease activity.
Cationic lipid reagent was oxidizedDo not vortex or agitate cationic lipid reagents excessively; this may form cationic lipid reagent peroxides.
Selection antibiotic added too soonWhen creating stable cell lines, allow at least 72 hr for cells to express the resistance gene before adding selective antibiotic.

Transfection results not reproducible

Possible cause(s)
Suggested solutions
Cells have changed over time, or splitting conditions have changedIf transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
Transfections performed at different cell confluencies, or at different DNA:transfection reagent ratios
Transfection performance reproducibility is dependent on day-to-day consistency in cell splitting, plating and transfecting with a consistent protocol (same DNA:transfection reagent ratios). Different DNA preparations or media changes may also change transfection performance.