6 replies to this topic

[WonderRakeWoman]

Hello everyone,

I am going to be making some doubly transfected cells with one plasmid that has neomycin resistance and one with puromycin resistance (it has an IRES site, which I've also never used before...). I have already made some singly-transfected cells with the plasmid that has Neo resistance and those are fine. I have never worked with the puro resistance marker and so I did a kill curve. The recommended puromycin concentrations I've seen are 1-10 ug/mL. When I did my kill curve though, I found that 0.25 ug/mL was enough to kill almost all of my cells (but 0.1 ug/mL was not enough to kill the cells)! Does anyone have experience or knowledge of another example where cells are killed by such low puromycin concentrations? I'm using Jurkat T cells.

Thanks!

[bob1]

if you can do a test on your neo selected cells to see if it works, in my experience the recommended ranges are bang on what works. I have never worked with T cells though.

[WonderRakeWoman]

I did the kill curve on both regular and neo selected cells and got the same result for both. I did a google search for "puromycin 250 ng/mL" and actually that concentration has been used in some papers (in other cell types)... so I guess I'll go with it if nobody else has any recommendations. Thanks for your input!

[dpo]

I have made some kill curves for quite some cell lines and 250 ng/ml is not that exceptional, I used it for several cell lines. So I wouldn't worry about it and just use the 250 ng/ml concentration.

[WonderRakeWoman]

Thanks dpo! I'm just glad to get some confirmation.

[vitalgene]

i used IRES vectors with puro selection on ESCs, what i would do with my cells is to start with 0,25 or 0,5 µg/ml puro for first two days, as the negative cells doesnt get killed overnight (but slowly) , that doesn't harm the transfected cells. and later increase the puro to 2µg/ml upto 4 µg/ml. If i start with higher puro ie >2, the negative cells are killed overnight and eventually harm the good cells. i tried this and found a big difference in obtaining the colony nummer at the end of selection process (day10)
I hope i was clear
good luck

[WonderRakeWoman]

Thanks, that sounds like a good idea. I think I will try dividing my cells in half and keeping 1/2 at 250ng and the other half I will try gradually increasing the puromycin.