Difference between revisions of "A1 cell transfection with FuGene"
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(Created page with "== Materials == * Fugene Transfection solution * Complete media for A1 cells * No serum media * ATE * APBS * 10 cm dishes * eppendorf tubes == Procedure == Incubation FuGen...") |
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| Line 1: | Line 1: | ||
| − | == | + | == Material == |
* Fugene Transfection solution | * Fugene Transfection solution | ||
* Complete media for A1 cells | * Complete media for A1 cells | ||
| Line 9: | Line 9: | ||
== 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) 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. | |
| − | cm dish. Downscaling or upscaling for different dish sizes are possible (ex. 6 µl for 6 well plates) | + | # 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: 5.1 Wash the cells with 5 ml APBS 5.2 Detach them wit ATE and stop the reaction with 5 ml HS – media 5.3 Pellet them by centrifuging them for 3 min @ 1000 rpm 5.4 Resuspend in complete media 5.5Seed the cells in 10 cm dishes | |
| − | each plasmid so that the sum of all the DNA in the solution is | + | # Add drop wise the FuGene – DNA mix (400 µl/ transfection), spread the cells homogenously in the plate |
| − | 6 µg | + | # Incubate the cells @ 25 degree 2% CO2 (after 2 days check) |
| − | |||
| − | 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. | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | 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: | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | Add drop wise the FuGene – DNA mix (400 µl/ transfection), spread the cells homogenously | ||
| − | |||
| − | Incubate the cells @ 25 degree 2% CO2 (after 2 days check) | ||
Latest revision as of 14:24, 8 June 2015
Material
- 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: 5.1 Wash the cells with 5 ml APBS 5.2 Detach them wit ATE and stop the reaction with 5 ml HS – media 5.3 Pellet them by centrifuging them for 3 min @ 1000 rpm 5.4 Resuspend in complete media 5.5Seed 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)