A1 cell transfection with FuGene: Difference between revisions
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== Procedure == | == Procedure == | ||
# Incubation FuGene – DNA: | |||
# 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) | 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 | NOTE: if the transfection is a cotransfection use amounts of | ||
each plasmid so that the sum of all the DNA in the solution is | each plasmid so that the sum of all the DNA in the solution is | ||
6 µg | 6 µg | ||
# 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. | |||
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. | # Incubate 20 min @ RT. | ||
Incubate 20 min @ RT. | |||
Cell preparation: | Cell preparation: | ||
Revision as of 11:27, 23 March 2015
Materials
- Fugene Transfection solution
- Complete media for A1 cells
- No serum media
- ATE
- APBS
- 10 cm dishes
- eppendorf tubes
Procedure
- Incubation FuGene – DNA:
- 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
- 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.
- Incubate 20 min @ RT.
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: - Wash the cells with 5 ml APBS - Detach them wit ATE and stop the reaction with 5 ml HS – media - Pellet them by centrifuging them for 3 min @ 1000 rpm - Resuspend in complete media - Seed the cells in 10 cm dishes
Add drop wise the FuGene – DNA mix (400 µl/ transfection), spread the cells homogenously in the in the plate
Incubate the cells @ 25 degree 2% CO2 (after 2 days check)