Educational guide
DNA Transfection Troubleshooting
DNA Transfection Troubleshooting Resources » Technical Resource Centers » Gene Technical Resources » Molecular Cloning Center » DNA Transfection Troubleshooting Molecular Cloning Strategies What is PCR Restriction Enzymes Digestion DNA Ligation Plasmid Transfo
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DNA Transfection Troubleshooting
Resources » Technical Resource Centers » Gene Technical Resources » Molecular Cloning Center » DNA Transfection Troubleshooting
Molecular Cloning Strategies
What is PCR
Restriction Enzymes Digestion
DNA Ligation
Plasmid Transformation
DNA Transfection Guide
Transfection Protocol
DNA Transfection Troubleshooting
Ligation Protocol
DNA Ligation Troubleshooting Guide
PCR Protocol, PCR Steps
PCR Troubleshooting Guide
Restriction Digest Protocol
Restriction Digestion Troubleshooting Guide
DNA Ligation Troubleshooting
What is DNA Transformation|Biology
DNA Transformation Protocol
Transformation Troubleshooting Guide
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.
Molecular cloning strategies
PCR
Restriction digestion
Ligation
Transformation
Transfection
Molecular cloning handbook
Bioinformatics tools
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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