stable cell line

1. Do a kill-curve to find out the lowest dose to kill your cells (7-10 days for G418 and 4-7 days for puromycin). Split your cells at at 1:10 for puromycin selection and 1:20 for G418 selection the day before the selection. Change the medium every 2 to 3 days. G418 from 100ug/ml to 1mg/ml. Puromycin use 0.1ug/ml to10 ug/ml.
2. Perform a transfection with the plasmid containing the drug selectable marker. Twenty-four hrs post-transfection, passage the cells (at 1:10 for puromycin selection and 1:20 for G418 selection) into fresh growth medium containing selective agent. A mock transfection should be performed in parallel as a control. Grow and passage the cells as necessary (usually 2-3 days), maintaining selection pressure by keeping the selective agent in the growth medium. After 1-2 weeks, a large number of the cells will be killed; the cells that remain growing in the selective medium have retained the expression plasmid, which stably integrates into the genome of the targeted cells. Monitor the mock control to ensure the cells are dying, no cells attached at the bottom.
3. If you pool all the colonies, that is your stable pool. If you isolate individual colony, that is your individual clones.