Difference between revisions of "Cloning of C2C12 cells"
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| + | 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 | ||
| + | |||
# | # | ||
Revision as of 12:28, 12 January 2015
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