Co-transfection ratio imbalance — uneven dual-plasmid expression (Transfection (Co-transfection))

Question

Accepted Answer

Of two co-transfected plasmids, only one expresses strongly; the fraction of double-positive cells is low. Common causes: 1) Plasmid sizes or promoter strengths differ significantly 2) DNA ratio between the two plasmids is wrong 3) siRNA / plasmid competition for cellular machinery 4) Different uptake efficiency between the two constructs 5) Cell type itself is hard to co-transfect efficiently Solutions: 1) Optimize plasmid size and quantity ratio (start with 1:1 by mass, then adjust by molarity) 2) Lower the total DNA amount — heavy DNA load suppresses overall expression 3) Test each plasmid alone to confirm individual transfection efficiency 4) For demanding experiments, consider a bidirectional vector or stable expression line