A1 cell transfection with FuGene: Difference between revisions
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# 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: 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. | # 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 | ||
Briefly: | |||
- Resuspend in complete media | - Resuspend in complete media | ||
- Seed the cells in 10 cm dishes | - Seed the cells in 10 cm dishes | ||
# Add drop wise the FuGene – DNA mix (400 µl/ transfection), spread the cells homogenously in the plate | # Add drop wise the FuGene – DNA mix (400 µl/ transfection), spread the cells homogenously in the plate | ||
# Incubate the cells @ 25 degree 2% CO2 (after 2 days check) | # Incubate the cells @ 25 degree 2% CO2 (after 2 days check) | ||
Revision as of 12:18, 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: 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
- Add drop wise the FuGene – DNA mix (400 µl/ transfection), spread the cells homogenously in the plate
- Incubate the cells @ 25 degree 2% CO2 (after 2 days check)