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Plasmid Transfection in 293T Cells: Principles and Hands-on Protocol

🚨 Failure Case Library (5) + Submit your own case

critical
Almost no fluorescence signal — transfection essentially failed
Under the fluorescence microscope, no GFP signal is visible; Western blot also shows essentially no target protein expression.
💡 5 · ✓ 5
severe
Bright fluorescence but Western blot shows very low expression
GFP fluorescence is obvious under microscope, but the corresponding band on Western blot is faint or near-invisible.
💡 5 · ✓ 5
moderate
Co-transfection ratio imbalance — uneven dual-plasmid expression
Of two co-transfected plasmids, only one expresses strongly; the fraction of double-positive cells is low.
💡 5 · ✓ 4
moderate
Bright at 24 h but signal rapidly decays at 48 – 72 h
Early time point shows strong fluorescence but it drops sharply within 48 – 72 h; signal varies dramatically between time points.
💡 4 · ✓ 4
minor
Orange Background Fluorescence with GFP
Light granular orange background fluorescence observed after GFP transfection with Lipofectamine 2000
💡 3 · ✓ 3
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