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  • Transfection Guidelines (E3314)

    Introduction

    IMPORTANT: 

    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:

    1. Transfect several 60 mM plates with a pCLuc-Basic 2-derived plasmid with an appropriate transfection agent for your cells.
    2. Replace the medium, return the cells to the incubator and continue with the incubation for another 24 hours.
    3. 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.).
    4. Replace the medium in the petri plates with the G418-containing medium and continue with the incubation for 24 hours.
    5. 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.
    6. Replace the medium before starting the isolation of colonies.
    7. Transfer colonies to a 96-well plate.
    8. 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.
    9. 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.
    10. Assay the CLuc activity in the supernatants to screen for positive clones.
    11. Expand the clonal cultures to the 48-well format.
    12. 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.