Very Low Transfection Efficiency (Lipid Transfection)

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Accepted Answer

Transfection efficiency is significantly lower than expected with poor gene expression Common causes: 1) DNA or transfection reagent diluted in media containing serum 2) Sub-optimal DNA to transfection reagent ratio 3) Insufficient or degraded plasmid DNA 4) Cell density not optimal (should be >90% confluent) 5) Promoter-enhancer not compatible with cell type 6) Inhibitors present in medium (antibiotics, EDTA, phosphate, sulfated proteoglycans) 7) Cells excessively passaged or splitting conditions changed Solutions: 1) Use serum-free medium (Opti-MEM) for dilutions before complexing 2) Optimize DNA:Lipofectamine 2000 ratio from 1:0.5 to 1:5 (typically 1:2 to 1:3) 3) Verify DNA concentration and quality using spectrophotometer or Quant-iT assays 4) Use high-quality plasmid DNA with PureLink HiPure purification kits 5) Ensure cell density >90% at transfection time 6) Use growth medium containing serum during transfection 7) Avoid antibiotics, EDTA, citrate, phosphate, RPMI, or sulfated proteoglycans 8) Use reporter gene to confirm expression 9) Start new vial of cells from liquid nitrogen if excessive passaging occurred