C2C12 cell transfection with FuGene
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2 x 6cm dishes with C2C12 cells
Material
Day | Material |
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first | 2x 6cm dishes
75 cm2 flask C2C12 cells in a 75 cm2 flask 15 ml FALCON tubes High serum media for C2C12 TE in PBS PBS Neubauer chamber for counting the cells Pipettes and tips |
second |
DMEM (4500mg/l Glucose) FuGene Transfection reagent the plasmid which should be transfected 1.5 ml eppendorf tubes high serum media for C2C12 pipettes and tips |
third |
Fresh prepared non-serum-T.i.- media PBS |
fourth |
PBS Fresh prepared 2% Horse Serum- low serum |
Procedure
Day | Procedure |
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first |
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second |
o 1) per transfection: 1µg plasmid in 40 µl DMEM o –for 2 transfections: 2µg plasmid in 80 µl o DMEM o pipette this in one 1.5ml eppendorf tube together 2) per transfection: 6µl of FuGene transfection reagent into 154 µl DMEM (total volume: 160µl) into a second 1.5ml eppendorf tube -for 2.5 transfections: 15 µl FuGene transfection reagent in 385 µl DMEM (in tube one should be now 400µl-> 200µl per transformation) [pipette at first the DMEM into the second tube and than into the DMEM the FuGene!] 3) pipette from the second tube (FuGene-DMEM-Mix) 320 µl of the Mix into the first tube (DNA-DMEM-Mix) 4) incubate the FuGene-DNA-DMEM-Mix for 15 min at RT
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third |
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fourth |
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