Hello Frank,

First thing to do before you transfect your cells is to do a dose-response curve of the effect of puromycin on your un-transfected cells viability. You want your selection with puromycin to be done within a week. So you should select a concentration of puromycin that would kill all untransfected cells in one week or less and leave only the transfected cells that will live and be stably transfected. The usual puromycin concentration used is 1-4 ug/ml depending on the cell line.

The selection (addition of puromycin to the cell culture media) is usually started around 24 hr following transfection. You can also start the selection at 48 hr depending on the health of your transfected cells.

You have to keep in mind that the protocol will vary if you want to keep the cells as a heterogeneous stable cell line or you want to pick single stably expressing colonies.

A week later all the living cells should be the stably transfected cells.

How long to keep the cells under selection. In some cases such as CRISPR-cas9 selection, the selection should be removed after 1 week. In other cases, puromycin is added at the selection concentration and then the cells are maintained in a lower puromycin concentration.

All works, it just depends on your cell line type and the experimental conditions.

Hello Frank,

First thing to do before you transfect your cells is to do a dose-response curve of the effect of puromycin on your un-transfected cells viability. You want your selection with puromycin to be done within a week. So you should select a concentration of puromycin that would kill all untransfected cells in one week or less and leave only the transfected cells that will live and be stably transfected. The usual puromycin concentration used is 1-4 ug/ml depending on the cell line.

The selection (addition of puromycin to the cell culture media) is usually started around 24 hr following transfection. You can also start the selection at 48 hr depending on the health of your transfected cells.

You have to keep in mind that the protocol will vary if you want to keep the cells as a heterogeneous stable cell line or you want to pick single stably expressing colonies.

A week later all the living cells should be the stably transfected cells.

How long to keep the cells under selection. In some cases such as CRISPR-cas9 selection, the selection should be removed after 1 week. In other cases, puromycin is added at the selection concentration and then the cells are maintained in a lower puromycin concentration.

All works, it just depends on your cell line type and the experimental conditions.

following

Hi Frank,

Here is a paper that shows doses of puromycin and time:

https://academic.oup.com/nar/article/46/9/4677/4972875

All the best!