protocol for selecting colonies in pX459 transfected cells

[regulatingge...@gmail.com]

Hello everyone,
I am wondering if someone has a written protocol for doing CRISPR in mammalian cell lines. I have transfected the pX459 (with puromycin resistance) and did selection for puromycin. I isolated the genomic DNA and did TIDE analysis on the polyclonal population of cells but the it did not seem that a big fraction of the cells had the endogenous locus KO. I want to point that I chose the top 3 gRNA from the MIT gRNA design tool. I was trying to avoid selecting colonies to save time. Does anyone have a protocol for selecting colonies? How long does the process take?
Thanks.

[Zach McLean]

I follow this procedure

http://www.farnhamlab.com/protocols/Farnham%20Lab_Puro%20CRISPR%20Protocol_20160115.pdf

[regulatingge...@gmail.com]

Hi Zach,
How often do you change the puro medium during the 2-3 week selection period?
Thanks

[jean-pierre de villartay]

be aware that, if I recall correctly, the first version of pX459 had an issue with the Puro gene. Adggene had communicated on this. I think the pX459-V2 is now ok
good luck

[Zach McLean]

To be clear, the selection with puro is transient in this case. As it says in the protocol, the day after transfection I add the puro and select for 48 hrs. You're trying to select on the transiently transfected cells to purify, not make cells a stably expressing the pX459 plasmid.

[regulatingge...@gmail.com]

Hi Zach,
So you do not add puromycin during the 2-3 week selection period of colonies in the 96 well plates?
Thanks.