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stable cell line - stable cell line (Feb/22/2002 )

I am looking for a good protocol on establishing a stable HeLa and RK13 cell line using a neomycin resistant plasmid?  Should the plasmid be linearized before transfection?  I need to obtain very high expression and therefore would like to obtain multiple insertional events.

-jeff langland-

I never linearized my DNA prior to transfection. For transcient and stable transfection, i use the product EXGEN 500 which yields high pourcentage of transfected cells. The DNA should be isolated with ethidium bromide, and uyou can try various amounts of transfected DNA. Use a control, such as luciferase, to view the relative percentage of transfection. Once I transfected, i wait 24 hours before i start treating with the antibiotic.(if it's the first time you use this antibiotic for transfection, it's better to test various concentrations on non-transfected cells to evaluate tehe best concentration to use, and to insure that the concentration is not to high). I treat my cells as long as needed to visualize clone formation in the wells. You can proceed with small cylindrical tubes coated with silicone, to add trypsin or viokase, to transfer those stable cells to 24 wells/plates, and start growing them. Once you have different stable clones, you can test every one to see which one posess the highest activity.