Materials
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 http://www.promega.com/a/apps/biomath/index.html?calc=dilution . Materials: Fresh complete media Trypsin 0.25% 6- 12- 24- well plates Incubator 37ºC—5%CO2 Jetprime buffer (Polyplus) Jetprime reagent (Polyplus) Plasmids Fluorescence microscope FACS
Methods
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 2º Transfection • 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
