Almost no fluorescence signal — transfection essentially failed (Transfection)

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Under the fluorescence microscope, no GFP signal is visible; Western blot also shows essentially no target protein expression. Common causes: 1) Plasmid quality is poor or partially degraded 2) Transfection reagent has lost activity or dose is wrong 3) Cells are in poor state (too dense, too sparse, or unhealthy) 4) Medium contains serum when the reagent requires serum-free transfection step 5) Protocol step was missed (e.g. complex not mixed adequately) Solutions: 1) Use high-purity plasmid (OD260/280 ≈ 1.8 – 2.0); avoid old preps 2) Optimize reagent and DNA amount ratio; check expiry 3) Use cells in log-phase growth at 70 – 80 % confluence 4) Use serum-free medium during the transfection step (follow reagent manual) 5) Strictly follow the protocol; mix the DNA-reagent complex thoroughly before adding