Low transfection efficiency
(1) Poor cell status or inappropriate cell density
It is recommended to inoculate cells with good growth status and inoculate cells at the optimal density required by the transfection reagent.
(2) Optimize transfection conditions
For example, optimize the amount of cationic liposome reagent and DNA.
(3) DNA-cationic liposome reagent complex is formed in the presence of serum
It is recommended not to use serum when the complex is formed.
(4) Whether there are inhibitors in the transfection system
It is recommended not to use antibiotics, EDTA, citrate, phosphate, RPMI, chondroitin sulfate, hyaluronic acid, dextran sulfate or other sulfated proteoglycans in the culture medium used to prepare DNA-cationic liposome complex.
(5) Freezing of cationic liposome reagent
It is recommended not to use frozen cationic liposome reagent or one stored at a temperature below 4°C.
(6) Problems with plasmid purification
It is recommended to use a transfection-grade plasmid purification kit.
High cell death rate
(1) DNA amount is too high
Recommendation: Perform a dose-response curve to determine the optimal amount of DNA. Add cationic lipid reagent to the dose-response transfection, because DNA alone will have a basal effect on cell growth.
(2) Cationic lipid reagent amount is too high
Recommendation: Perform a dose-response curve to determine the optimal amount of cationic lipid reagent. Add DNA to the dose-response transfection, because cationic lipid reagent alone will have only a basal effect on cell growth.
(3) Antibiotics used during transfection
Recommendation: Do not use chloramphenicol, penicillin, or streptomycin during transfection, because cationic lipid reagents make cells more sensitive.
(4) Too few cells
Recommendation: Perform a dose-response curve to determine the optimal number of cells per transfection. Adjust the number of cells according to the efficiency required for your application.
(5) Cationic lipid reagent oxidized
Recommendation: Do not excessively agitate or shake the cationic lipid reagent, which may form peroxides of the cationic lipid reagent.
(6) For stable transfection, the selection antibiotic was added too quickly or at too high a concentration
Recommendation: Before adding the selection antibiotic, the cells should be cultured in a medium without G418 for at least 48 hours to allow the cells to express the resistance gene.
Poor transfection reproducibility
(1) Fluctuation of confluency during transfection
Recommendation: Keep all transfection parameters constant for different batches, such as confluency, number of passages, and growth time.
(2) Cell changes during culture
Recommendation: If possible, use cells from a sublineage that has been selected for higher transfection efficiency. Perform experiments with freshly thawed cells.