Real lab failures, root causes, and fixes — curated and bilingually annotated by our team.
Fluorescence forms aggregates inside cells, or localizes to a compartment different from the expected native pattern.
GFP fluorescence is obvious under microscope, but the corresponding band on Western blot is faint or near-invisible.
Cells become rounded and detached after transfection; CCK-8 shows a sharp drop in viability vs. mock-transfected control.
Microscopy shows scattered positive cells; flow cytometry confirms positivity rate is very low (< 5 %).
Under the fluorescence microscope, no GFP signal is visible; Western blot also shows essentially no target protein expression.
Early time point shows strong fluorescence but it drops sharply within 48 – 72 h; signal varies dramatically between time points.
Parallel wells show very different fluorescence intensity / positive-cell rates; data fluctuates wildly between replicates.
Untransfected or empty-vector wells show a clearly green/red signal — you can't tell if the target plasmid is really expressing.
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