NIH3T3 transfection with Jetprime Polyplus

Posted by jmmormoy

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
Trypsin 0.25%
6- 12- 24- well plates
Incubator 37ºC—5%CO2
Jetprime buffer (Polyplus)
Jetprime reagent (Polyplus)
Fluorescence microscope


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