hi depending upon the system you are using single plasmid (sg+cas9), two plasmid ...i am using successfully thetwo plasmid system on with the guide one with the cas9-puro..transfect yoir cells, the next day start selecting for puro resistance, when the controls have died, trypsin the cell 1) plate 1000cells in a 10cm dish (without puromycine) and 2) seed cell on coverslip 6wells plate .;grow these cells...then take out the CS and fix teh cells, process the remaining cell monolayer for westen blot

perform the wb and IF analysis of the CS to assess the KO efficiency.;this will tell you the extend of KO...(with some system the KO can be as high as 90%)

let the cells grow in the 10cm plate and monitor the grow of the single cells...depending of the KO efficiency, collect the clone with cloning whatman paper (itis best that somebody show you this if possible, shortly, locate the colony you want to pick (as much as you can) wash the cell monolayer with pbs , drain the liquid off the plate, dip the whatman paper in trypsin, and overlay one disk over a colony, monitor the cell detachment which are visible under teh scope..one acheived, with the forceps, take out the whatman sterile and put it in a 96well plate, with medium, growth for 2-3aday, then remove the whatman with sterile forceps, growth the cells and expand for wb /IF analysis.

it is easy ..to perform

today there are several clever way to counter select the Ko

have a look

https://www.nature.com/protocolexchange/protocols/5845

hope this help

see also https://www.researchgate.net/post/Does_anyone_have_some_experience_useful_advice_for_isolating_single_clones

Hi Dr. Fesquet,

Thanks for the time and suggestions. I wish, I had used a plasmid with antibiotic resistance. Currently, I am using plasmid with OFP marker. The filter paper based isolation looks super helpful. I will try that. My challenge was getting single clone and I am hoping to get some clones with KO.

Thank you very much.