C2C12 cell transfection with FuGene: Difference between revisions
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385 µl DMEM (in tube one should be now 400µl-> 200µl per | 385 µl DMEM (in tube one should be now 400µl-> 200µl per | ||
transformation) | transformation) | ||
[pipette at first the DMEM into the second tube and | |||
than into the DMEM the FuGene! | 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) | 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 | 4) incubate the FuGene-DNA-DMEM-Mix for 15 min at RT |
Revision as of 12:58, 3 November 2014
2 x 6cm dishes with C2C12 cells
Material:
Day | Material |
---|---|
1 | 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 |
2 |
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 |
3 |
Fresh prepared non-serum-T.i.- media PBS |
4 |
PBS Fresh prepared 2% Horse Serum- low serum |
Procedure:
Day | Procedure |
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1 |
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2 |
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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3 |
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4 |
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