Cloning of C2C12 cells: Difference between revisions
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== | == Material == | ||
* PBS | * PBS | ||
* TE | * TE | ||
| Line 10: | Line 10: | ||
* 96 well plates (or another kind of well plates for the cloning → normally start with 96 well plates) | * 96 well plates (or another kind of well plates for the cloning → normally start with 96 well plates) | ||
== Procedure | == Procedure == | ||
# remove 5 ml of the old media in a 15 ml FALCON tube | # 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!!) | |||
96/ 62,5 cells = 1,5 µl of the cell suspension in 9.6 ml media | # 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 | |||
IT WILL BE A GOOD CLONING IF ~50% OF THE WELLS ARE EMPTY!!!!! | |||
Latest revision as of 14:32, 8 June 2015
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
- 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