Use good quality plasmid DNA, i.e., CsCl or standard maxiprep. Do not use mini-prep DNA.
Use proliferating mammalian cultures, i.e., regularly passaged cells.
Use complete growth medium without antibiotics and antimycotics to plate cells for transfection.
Use pSV40-CLuc to establish the transfection efficiency in a particular cell line.
Titrate the amount of plasmid and transfection reagent to achieve optimal transfection efficiency.
Protocol for Creating Stable Cell Lines:
- Transfect several 60 mM plates with a pCLuc-Basic 2-derived plasmid with an appropriate transfection agent for your cells.
- Replace the medium, return the cells to the incubator and continue with the incubation for another 24 hours.
- Start the selection by preparing growth media containing a range of G418 concentrations (e.g. 250 μg/ml, 500 μg/ml, 1 μg/ml etc.).
- Replace the medium in the petri plates with the G418-containing medium and continue with the incubation for 24 hours.
- Continue to replace the medium daily until the majority (~99%) of cells are dead, then stop replacing the medium and allow the plate to incubate (for ~7–10 days) until colonies begin to appear.
- Replace the medium before starting the isolation of colonies.
- Transfer colonies to a 96-well plate.
- Replace the medium in the petri plate before returning it to the incubator. Cells can be transferred to 96-well plates until colonies begin to merge on the petri plate.
- Replace the medium for the isolated clones every 2-3 days until the cell density reaches ~80%.
Note: Use medium containing the concentration of G418 from which the colonies were isolated.
- Assay the CLuc activity in the supernatants to screen for positive clones.
- Expand the clonal cultures to the 48-well format.
- Repeat the expansion (to 24-well, to 12-well, to 6-well plates, to a T25 flask and etc.) until sufficient cell stock has been generated for freezing.