Difference between revisions of "C2C12 cell transfection with FuGene"
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''2 x 6cm dishes with C2C12 cells'' | ''2 x 6cm dishes with C2C12 cells'' | ||
− | == Material | + | == Material == |
{| class="wikitable" | {| class="wikitable" | ||
!style="width: 10px" | Day | !style="width: 10px" | Day | ||
!style="width: 590px" | Material | !style="width: 590px" | Material | ||
|- | |- | ||
− | | | + | |first |
| 2x 6cm dishes | | 2x 6cm dishes | ||
75 cm2 flask | 75 cm2 flask | ||
Line 17: | Line 17: | ||
Pipettes and tips | Pipettes and tips | ||
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− | | | + | |second |
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DMEM (4500mg/l Glucose) | DMEM (4500mg/l Glucose) | ||
Line 26: | Line 26: | ||
pipettes and tips | pipettes and tips | ||
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− | | | + | |third |
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Fresh prepared non-serum-T.i.- media | Fresh prepared non-serum-T.i.- media | ||
PBS | PBS | ||
|- | |- | ||
− | | | + | |fourth |
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PBS | PBS | ||
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|} | |} | ||
− | == Procedure | + | == Procedure == |
{| class="wikitable" | {| class="wikitable" | ||
!style="width: 10px" | Day | !style="width: 10px" | Day | ||
!style="width: 590px" | Procedure | !style="width: 590px" | Procedure | ||
|- | |- | ||
− | | | + | |first |
| | | | ||
− | + | # wash the cells with 5 ml PBS | |
− | + | # add 2 ml of TE to the cells and incubate them for 2 min at 37ºC | |
− | + | # during this time: label the new flask and the 2 6cm dishes with the name and the passage number of the cells and the date and fill in the flask 15 ml of new media | |
− | + | # stop the TE with the 5 ml of the HS – media | |
− | + | # transfer the cell suspension into the 15 ml FALCON tube | |
− | + | # centrifuge the cells: 1000xg for 3 min at +4ºC | |
− | + | # remove the old media and dissolve the cell pellet in a few ml of fresh media (the amount of media correlates with the pellet- the cells should be not to dense or to diluted for the counting – general 3 ml are fine for a pellet of one 75cm2 flask of C2C12) | |
− | + | # prepare the Neubauer camber (put the cover slip over the chamber and move it a little bit till you can see the Newton’s rings!!! – this is important for the calculation!!) | |
− | + | # fill between the camber and the cover slip around 40 ml of cell suspension | |
− | + | # count the cells under the scope (all 4 corner crosses - each corner cross has 16 little crosses)- count the both main crosses | |
− | + | # to calculate the number of cells which are in (f.ex. in total 3mls) suspension, calculate at first the average of the 8 crosses, than multiply the average with 104 (the number of cells in 1 ml (!) of suspension) and than multiply this with the amount of ml of the suspension (f.ex. 3) – this is the total number of cells | |
− | + | # pipette in each 6 cm dish 350.000 cells 9next day: use this for transfection) and in the flask 1:30 | |
− | + | # 6 cm dishes: fill up to total 5 ml with fresh media | |
− | + | # move the dishes like a cross that the cells are homogenized in the dish | |
− | + | # let them grow at 37ºC, 10% CO2 | |
|- | |- | ||
− | | | + | |second |
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− | + | # FuGene transfection reagent (stored at –20ºC) and plasmid on ice | |
− | + | # For 2 transfections: | |
− | o 1) per transfection: 1µg plasmid in 40 µl DMEM | + | o 1) per transfection: 1µg plasmid in 40 µl DMEM |
− | o –for 2 transfections: 2µg plasmid in 80 µl | + | o –for 2 transfections: 2µg plasmid in 80 µl |
− | o DMEM | + | o DMEM |
− | o pipette this in one 1.5ml eppendorf tube together | + | o pipette this in one 1.5ml eppendorf tube together |
− | 2) per transfection: 6µl of FuGene transfection reagent into 154 | + | 2) per transfection: 6µl of FuGene transfection reagent into 154 |
µl DMEM (total volume: 160µl) into a second 1.5ml eppendorf tube | µl DMEM (total volume: 160µl) into a second 1.5ml eppendorf tube | ||
-for 2.5 transfections: 15 µl FuGene transfection reagent in | -for 2.5 transfections: 15 µl FuGene transfection reagent in | ||
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 |
− | + | # during this time: remove the media from the cells and add in each dish 1.8 ml of DMEM- put this for the rest of the incubation time at 37ºC (incubator) | |
− | + | # after the 15 min take the cells put from the incubator and drop wise pipette into each dish 200µl of Fugene-DNA-DMEM-Mix | |
− | + | # incubate this for one hour at 37ºC (incubator) | |
− | + | # after this: ADD 4 ml of high serum media to each dish | |
− | + | # dishes back in the incubator (37ºC, 10%CO2) | |
|- | |- | ||
− | | | + | |third |
| | | | ||
− | + | # if all wash fine, than the cells should be now confluent | |
− | + | # if this is the case: remove the media, wash the cells twice with PBS and add 5 ml of Non-serum-T.i.-medium to each dish | |
|- | |- | ||
− | | | + | |fourth |
| | | | ||
− | + | # remove the media and wash the cells twice with PBS | |
− | + | # add 5 ml of 2% HS-LS to each dish | |
|} | |} |
Latest revision as of 14:32, 8 June 2015
2 x 6cm dishes with C2C12 cells
Material
Day | Material |
---|---|
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 |
---|---|
first |
|
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
|
third |
|
fourth |
|