Lipofectamine® 2000 Transfection Reagent

Protocol tips

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	Add diluted DNA to diluted Lipofectamine® 2000 Reagent and incubate for 5 minutes at room temperature. Incubate cells for 18h at 37°C and change medium and re-incubate for 72 hours

Protocol tips
Add diluted DNA to diluted Lipofectamine® 2000 Reagent and incubate for 5 minutes at room temperature. Incubate cells for 18h at 37°C and change medium and re-incubate for 72 hours

Publication protocol

HIESC-2 (Chapdelaine et al., 2006) were cultured in 6-well plates and maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and penicillin–streptomycin 1× (complete medium) in a 37°C humidified incubator with 5% CO2. When the cells reached 80% confluence the complete RPMI medium of each well (3 ml) was replaced with 2 ml of fresh RPMI medium without antibiotic. Co-transfection of the cells was done as follows: for each well, 4 μg of plasmid expressing the different sgRNAs (identified as sgRNA target #14, 15, 16 and 17) (see Fig. 2B) and 2 μg of the Cas9 plasmid (Addgene, Cambridge, MA, USA cat no 48668) diluted in the Opti-MEM medium were mixed with 10 μl of lipofectamine diluted in Opti-MEM medium. The complexes were added to each well and incubated for 18 h at 37°C before medium replacement with complete RPMI. Three days after transfection, cells were harvested and genomic DNA was analyzed for the presence of mutation using the Surveyor® enzyme as described below. At the same time, since both plasmids carry the neomycin resistance gene, transfected cells were deemed overrepresented among resistant clones. A sample of the cells were cultured in the presence of neomycin (400 μg/ml) for 2 days followed by two additional days in complete medium without neomycin after which cells were trypsinized, diluted and plated in 6-well plates. After 10 days, individual colonies were isolated with O-rings, harvested, seeded in 24-well plates and grown for 2 weeks until confluence when they were transferred to T25 flasks. When the latter reached confluence, cells were trypsinized and an aliquot was kept for protein analysis by western blot. The remaining cells were frozen or expanded in T75 Flasks for additional analyses.

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