cotransfect px461 and px462?

[LiHD]

Hello,

Do you think I could put one gRNA in px461(Cas9n-GFP) and one gRNA in px462 (Cas9n-Puro) and cotransfect them cells, so that I could use both GFP and Puro for postive cell selection?

Thanks!

HD 

[Fatwa Adikusuma]

Why not, I think that's the best way to do it. We normally do puro selection. If you use puro selection, I recommend you to transfect higher amount of px461 compared to px462. I used to co-transfect 2 CRISPR plasmid (one in PX459puro, another one in px330 or px458) to cells with a very high efficiency in getting clones hit by both CRISPR. The reason I put one CRISPR in PX459-Puro and another one in Cas9-GFP/Cas9-only is because we wanna  make sure both plasmids enter the cells. I will give you an example, if we put both plasmid in PX459-Puro (let's say A & B), after puro selection the surviving clones must contain PX459-puro, but the surviving clones might contain only A or only B, and could be both.

However if we put CRSIPR A in PX459-puro, and CRISPR B in PX458-GFP/PX330 with ratio A:B=1:2 or even 1:3, after the puro selection the surviving cells must contain PX459-puro CRISPR A, and because CRISPR B in PX458-GFP/PX330 is much higher concentrated, the probability of CRISPR B to enter the cells together with CRISPR A is high. Thus we are convinced CRISPR A & B are transfected together to the cells

Cheers,

Fatwa

[LiHD]

Dear Fatwa,

Thank you very much for your answer! It's real helpful!

H-D

[Cho Rattanasinchai]

Hi HD,

I wonder if you have tried co-transfect cells with these two plasmids already?  I want to try the same transfection too but I am worried about the viability of the cells after GFP sorting.

Thanks,

Cho

[Tim]

Hello everyone,

Did one of you tried a co-transfection of PX458/459 or PX461/462? What do you think is the best way to select positive clones? First puro selection, then FACS or the other way round?

Thanks a lot!

Tim