Re: How long it takes for puromycin selection

[Neville Sanjana]

Hi Jerry,

We normally add puro 12-24 hours after transfection (for lentiviral infection, we wait 24 hours). For HEK293FT cells, puro 1ug/ml should be sufficient for selection and control cells (not puro resistant) should be nearly totally dead by 24 hours after puro addition. Your dilution strategy sounds fine to me.

Hope that helps,

Neville

On Mon, Oct 5, 2015 at 4:04 PM, Jerry Xu <gerryb...@gmail.com> wrote:

Hi guys

After transfection of Cas9 plasmid I started to add puromycin at 2ug/ml concentration for Hek293FT

Now it's been 18-19 hours but I don't see any change of cell morphology. Even for control cells (without transfection so without puromycin resistance) they seem to live happily.

So I'm wondering how long it takes to see the cell death (at least for control cells). What does the "death" looks like?

As for puromycin dilution, I directly add 2ul of 10mg/ul puromycin to 10ml culture medium for 5000 dilution, is it OK?

Thx

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[Jerry Xu]

Thx

I think my problem is I plated too many cells.

How much HEK293FT you plated for 24-well or 12-well?  I didn't know they grew so fast. 

[Neville Sanjana]

Yes, the FT cells grow amazingly fast! For effective puro selection, cells need to be on the sparser side.

For lentiviral infection, I typically infect ~25-50K cells in a 24 well dish but they can also be split before/during puro if needed.

For transient transfection, the cells will be more delicate after transfection. So, for transfection, I would transfect them at 80-90% confluence, remove the transfection mix after 6 hours and replace with normal media (this will make them healthier post-transfection)..... then you can passage if they are too dense before starting puro selection at 12+ hours after transfection.

You might need to optimize your strategy but as long as you have puro sensitive cells (I often infect/transfect with a EGFP virus/plasmid that doesn't have puroR), it will be easy to tell when selection has been effective.

Best,

Neville