Plasmid DNA, siRNA, or transfection reagent was diluted in serum-containing medium or formed complexes in the presence of serum.

Use serum-free medium to dilute plasmid DNA, siRNA, and transfection reagent.

The dilution time or complex incubation period was too short.

We recommend diluting DNA and transfection reagent separately and incubating for 5 minutes.  Then, combine the diluted reagents and incubate for 20 minutes for optimal performance.

The transfected plasmid DNA or siRNA was degraded or of poor quality.

Ensure that the plasmid DNA or siRNA used for transfection is of high quality.

There are inhibitors present in the culture medium.

Do not use antibiotics, EDTA, citrate, phosphate, RPMI, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the growth medium or the medium used to prepare the DNA:transfection reagent complex.

Problems exist in the assay used to measure efficiency or expression.

Use a reporter gene to measure transfection efficiency.

The cells cannot recognize the promoter/enhancer on the vector.

Verify that the promoter/enhancer on the vector construct is compatible with the target cell type.

The cells have changed over time, or the splitting conditions have been altered.

If transfection performance suddenly declines, it may be related to the cells.  We recommend splitting and plating cells on a consistent schedule and maintaining a cell density that is neither too sparse nor too confluent.  Over-passaging can also reduce transfection performance.

After adding the transfection complexes to the medium, ensure thorough mixing across the entire plate or well.

Avoid excessive vortexing or agitation of cationic lipid reagents, as this may promote the formation of lipid peroxides.

When establishing stable cell lines, allow at least 72 hours of resistance gene expression before adding selective antibiotics.