Transfection protocols vary based on cell type to be transfected, transfection method, and transfection reagent. The common causes of transfection failure are decrease transfection efficiency and decreased cell viability. Use the table below to troubleshoot transfection experimental failure. Ensure the quality of your target construct by getting the desired gene in the vector you want the easy way with GenEZ™ ORF Clones. Start with a Search for your gene.

Problem Cause Solution
Low transfection efficiency
DNA concentration too low or DNA is degraded
  • Increase ratio of DNA (µg):transfection reagent (µl)
  • Confirm DNA integrity by A260/A280 spectrophotometer reading (should be at least 1.7)
  • Perform gel electrophoresis to confirmed % of nicked DNA is less than 20%
Complexes not properly formed
  • Omit serum from complex formation step, use serum free media for DNA dilutions
  • Increase complexing reaction incubation time
Culture medium contains contaminants
  • Do not use antibiotics at time of transfection
  • Test cells for mycoplasma, yeast or other contaminants
Cells have suffered mechanical damaged 
  • Do not vortex or spin cells for extended period of time
Transfection reagent has been compromised
  • Some transfection reagents can be compromised via freezing or storage long tem at room temperature. Store reagents at 4°C
Low cell viability after transfection
DNA used for transfection was contaminated
  • DNA may contain contaminants
Target cells are compromised
  • Use low-passage-number-cells (less than 20) or use fresh vial of cells
  Cell density too low
  • Ensure that cells are 70 to 90% confluent at time of transfection


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