Cloning of C2C12 cells

Material

• PBS
• TE
• 1 FALCON tube (15ml)
• HS for C2C12
• Neubauer Chamber
• the same amount of white tubes like plates to prepare
• pipettes and tips
• Multipipette and 5 ml tips
• 96 well plates (or another kind of well plates for the cloning → normally start with 96 well plates)

Procedure

1. remove 5 ml of the old media in a 15 ml FALCON tube
2. through the rest of the old media away
3. wash the cells carefully with PBS
4. remove the PBS
5. add 2 ml TE to the dish and let it incubate till all cells are detached (~ 4 – 5 min)
6. stop the TE with the old media and resuspend the cells in the old media -> pipette the suspension in the FALCON tube back
7. pellet the cells (1000 xg, 3 min, 4ºC)
8. remove the old media
9. resuspend the cells in new media (incase how many cells you think to have) -> normally between 3 ml till 10 ml
10. resuspend the cells very well
11. put the cover slip over the Neubauer Chamber and fill between the both with a pipette the cell suspension
12. count the cells two times in the corner crosses (4 pieces per cross -> two crosses per chamber)
13. calculate the average -> multiplied the result with 104 and this number of cells means: cells/ ml
14. in each well (96 well plate) use as a total volume 100 µl → this means that if you want to have 1 cell per well than you should calculate the dilution that you have 96 cells in 9.6 ml media
15. for example: 62500 cells/ ml = 62.5 cells/ µl (96/ 62,5 cells = 1,5 µl of the cell suspension in 9.6 ml media)
16. if you want to prepare 5 * 96 well plates, than take 5 white tubes (take the lid away) and pipette in each tube 10 ml media
17. add in each tube (calculation from the example) 1.5 µl cell suspension (before resupend the cells very well!!)
18. take a MULTIpipette and a 5 ml tip (use for each plate a new tip)
19. arrange the pipette to 100 µl, resuspend the cells with the MULTIpipette very well
20. pipette in each well 100 µl of the cellsuspention (IT WILL BE A GOOD CLONING IF ~50% OF THE WELLS ARE EMPTY!!!!!)
21. fill the rest of the cells in a 6 well plate with different concentrations