For cotransfection in this particular cell line we will be using “Jetprime” (Polyplus) reagent that has shown the best transfection efficiency compared to others (80% transfection with the control plasmid, check Notebook 198 page 53).
Also it is important to include control gRNA targeting a different locus to determine if there is non-homologous integrations.
For cotransfection use equimolar concentrations of hCas9 : Reporter : gRNA : donorDNA
Fresh complete media
6- 12- 24- well plates
Jetprime buffer (Polyplus)
Jetprime reagent (Polyplus)
1º Cell seeding (60-80% confluency) (Better transfection when seeding the same day as transfection).
• Choose the type of plate to transfect and seed experiments in replicates for checking transfection efficiency:
24 well — 50,000-80,000 cells — 0.5mL Media
12 well — 75,000-150,000 cells — 1mL Media
6 well — 150,000-250,000 cells — 2mL Media
• Dilute DNA on Jetprime buffer and vortex 10’’:
24-well — 50uL buffer — 0.5ug totalDNA
12-well — 100uL buffer — 1ug totalDNA
6-well — 200uL buffer — 2ug totalDNA
• Add jetprime reagent and vortex 10’:
24-well — 1uL Reagent
12-well — 2uL Reagent
6-well — 4uL Reagent
• Incubate mix 10’ – Room Temperature
• Add transfection mix to the cells in serum containing medium.
• Incubate for 48h at 37ºC—5%CO2
• Renew media after 48h and check fluorescence of reporter plasmid under microscope
3º Check transfection efficiency by FACS