1. Do a kill-curve to find out the lowest dose to kill your cells (4-7 days for puromycin). You can do that any time. You just need to know what concentration to use to select for your stable transfected clones.
Split your cells at 1:10 the day before the selection. Change the medium with different concentration of puromycine every 2 to 3 days. The normal conc. used for Puromycin is from 0.1 ug/ml to 10 ug/ml. So you can pick up a series of conc. within this range. For example, you can use 6 well plates, with each well containing a different conc. of puromycin (0, 0.1, 0.5, 1, 2, 10). You can pick more point if you want, so that you can get more accurate data. Check your cells every day, after 1 week, find the lowest dose that kill all your cells (100%). That’s the concentration you want to use for selection.
2. Perform a transfection with the plasmid containing Puro resistant gene. Twenty-four hrs post-transfection, passage the cells at 1:10 into fresh growth medium containing puromycin at the concentration you found from step 1. 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 puromycin 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. For isolation of individual colony, there are several commercial tools to do that. You can also keep on diluting your stable transfected cells to the point that you can assume every well only get one colony. Continue growing these cells in selection medium for 1-2 additional passages. At this time, each well contains a clonal population of stably transfected cells, which can be maintained in normal growth medium without the selection pressure of puromycin (although you may wish to grow the cells under “light pressure”, lower concentration of puromycin). These populations can be used for experiments or stored under liquid nitrogen in growth medium with 10% DMSO and 20% FBS for future use.