Concentration of puromycin in KO cells selection

[yoyodin...@gmail.com]

Hi all,

I have an question about the puromycin concentration in KO cells selection.Generally we make a killing curve so we will know the concentrations to kill all cells in two days or 7 days.Since we use PX459 to transient express Cas9 protein and were generally recommend to select for no more than 48h with puromycin.What concentration I should use for 48h selection?Use higher concentration which can kill all cells within 48h,or use lower concentration which can kill them in 7 days  but only maintain for 2 days?

In my experience,the 48h killing concentration is generally 2-3 folds higher than 7days killing concentration.I have tried use high concentration (5ug/ul) to select for 48h,however neither control cells nor transfected cells  can survive under this concentration.I plan to use 7days killing concentration for selection but I am afraid this low concentration can not kill all untransfeted cells within 48h.

Do you have any experience or suggestions on this problem?Any suggestion or discussion will be greatly appreciated!

My best,

Yao

[Vikas Daggubati]

Hi Yao, 

What cell line are you using? In my experience puromycin selection works best in 72 hours and for example, RPE1 cells use 5µg/mL in my experience. 

Best, 

Vikas