Cloning of C2C12 cells

Materials:

- 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:

- remove 5 ml of the old media in a 15 ml FALCON tube - through the rest of the old media away - wash the cells carefully with PBS - remove the PBS - add 2 ml TE to the dish and let it incubate till all cells are detached (~ 4 – 5 min) - stop the TE with the old media and resuspend the cells in the old media -> pipette the suspension in the FALCON tube back - pellet the cells (1000 xg, 3 min, 4ºC) - remove the old media - resuspend the cells in new media (incase how many cells you think to have) -> normally between 3 ml till 10 ml - resuspend the cells very well - put the cover slip over the Neubauer Chamber and fill between the both with a pipette the cell suspension - count the cells two times in the corner crosses (4 pieces per cross -> two crosses per chamber) - calculate the average -> multiplied the result with 104 and this number of cells means: cells/ ml

- 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

- 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

- 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 - add in each tube (calculation from the example) 1.5 µl cell suspension (before resupend the cells very well!!) - take a MULTIpipette and a 5 ml tip (use for each plate a new tip) - arrange the pipette to 100 µl, resuspend the cells with the MULTIpipette very well - pipette in each well 100 µl of the cellsuspention

IT WILL BE A GOOD CLONING IF ~50% OF THE WELLS ARE EMPTY!!!!!

- fill the rest of the cells in a 6 well plate with different concentrations