Transfections Not Reproducible (Lipid Transfection)

Question

Accepted Answer

Transfection efficiency shows high variability between experiments and among replicates Common causes: 1) Cells changed over time or splitting conditions inconsistent 2) Transfections performed at different cell confluencies 3) Different DNA:transfection reagent ratios used 4) Multiple pipetting errors when preparing complexes separately 5) Carryover from re-used tips especially in 96/384-well formats Solutions: 1) Keep all transfection parameters consistent: cell confluency, passage number, growth phase 2) Split and plate cells on consistent schedule, never too sparse or too dense 3) Thaw fresh cells if possible 4) Prepare one master mix of DNA/lipid complexes for all transfections 5) Change tips between wells to avoid carryover 6) Co-transfect internal normalization control (beta-galactosidase or luciferase) 7) Start new vial from liquid nitrogen if excessive passaging occurred