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  
[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
1
  • 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
2
  • FuGene transfection reagent (stored at –20ºC) and plasmid on ice
  • For 2 transfections:
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
  • 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)
3
  • 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
4
  • remove the media and wash the cells twice with PBS
  • add 5 ml of 2% HS-LS to each dish