Protocol provided by the MISSION® team
The appropriate concentration of antibiotic for selecting stable cell lines is different for each cell type. If the concentration for the desired cell type is unknown, a titration experiment must be performed to determine the lowest concentration of antibiotic needed to efficiently select transduced cells. In this protocol, we highlight the use of puromycin, the antibiotic utilized with the standard pLKO.1 vector. Typically, 1-10 µg/mL are sufficient to kill most untransduced mammalian cell types. This general protocol can also be utilized for determining the optimal concentration of G418 required for the custom shRNA vectors. Higher concentrations than absolutely required of antibiotic could lead to off target effects and fewer cells for downstream analysis.
If cells started to round but did not detach from the plate surface after addition of puromycin: