I need the procedure of how to establish a stable cell line urgently.
Dear Joshua,
In my case, I used Huh7 to establish a stable cell line expressing my interested protein. 24 h before transfection, I seeded the cells on 60 mm dishes. The day after, I performed transfection as usual (I used PEI for transfection and changed media 4 h after the transfection). 24 h later, cells were trypsinized and about 1/5-1/8 of the cells were plated into a 100 mm dish. G418 were treated with a concentration of 500 ug/mL (You should check how much G418 can kill your cell line). I changed the media every 2 day and supplemented with the G418 same as above. About 1 week from the treatment, cells will begin to die. 3-4 weeks after the treatment, you can see the colony by naked eye (like some acnes on the dish). Then you can wash the dish by PBS, used 20 ul pipette to add about 12 ul of trypsin onto the colony, pipetting up and down many times to collect all the colony and plating them into a new 12-well plate. You grow them up and verify the colony by immunoblot analysis (using the antibody against your interested protein or the tag).
Good luck!
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