A1 cell transfection with FuGene

Materials

• Fugene Transfection solution
• Complete media for A1 cells
• No serum media
• ATE
• APBS
• 10 cm dishes
• eppendorf tubes

Procedure

1. Incubation FuGene – DNA:
2. 18 µl FuGene: 6 µg DNA if the transfection has to be done in 10 cm dish. Downscaling or upscaling for different dish sizes are possible (ex. 6 µl for 6 well plates) NOTE: if the transfection is a cotransfection use amounts of each plasmid so that the sum of all the DNA in the solution is 6 µg
3. The Protocol is the same suggested from the manufacturer: prepare DNA in the appropriate number tubes in A1 media without serum (200 µl/ tube). In parallel prepare a master mix of FuGene in no serum A1 medium (200 µl each transfection), incubate the mix 5 min @ RT than add the FuGene containing medium to the DNA containing tubes.
4. Incubate 20 min @ RT.
5. Cell preparation: In the meantime prepare the cells: for one 10 cm dish transfection we need one 175 cm flask, detach and seed into a 10 cm dish, following the same protocol we used for passage A1 cells. Briefly: 1. Wash the cells with 5 ml APBS 2. Detach them wit ATE and stop the reaction with 5 ml HS – media 3. Pellet them by centrifuging them for 3 min @ 1000 rpm

- Resuspend in complete media - Seed the cells in 10 cm dishes

1. Add drop wise the FuGene – DNA mix (400 µl/ transfection), spread the cells homogenously in the plate
2. Incubate the cells @ 25 degree 2% CO2 (after 2 days check)