Electroporation of C2C12 cells: Difference between revisions
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== | == Material == | ||
* 1x Steinberger Medium | * 1x Steinberger Medium | ||
* 1x DMEM without serum | * 1x DMEM without serum | ||
== Procedure == | |||
- remove the old media | - remove the old media | ||
- wash the cells with 5 ml PBS | - wash the cells with 5 ml PBS | ||
Latest revision as of 14:33, 8 June 2015
Material
- 1x Steinberger Medium
- 1x DMEM without serum
Procedure
- remove the old media - wash the cells with 5 ml PBS - remove the PBS - add 2 ml TE - put the cells nearly 4 min in the 37 ºC incubator → for 6 transfections
- 6 * 6 cm dishes - label those with the Plasmid name
- cells from the incubator: - stop the TE with HS – media - resuspence the cells in the old media - remove the cells in the FALCON flask - spin the cells: 1000 rpm; 3 min
- label the electroporation cuvetts: 1 – 6 - after spinning: remove the old media in the waste - clean the pellet carefully with PBS ⇒ don’t resuspend the cells - remove the PBS - resuspend the cells in 1,8 ml media (for each cuvette: 300 µl) (very well resuspending) - pipette in each cuvette 300 µl from the cellsuspendion - add in the cuvette the right plasmid - DNA – c.: 0,05 µg/ ml - closed he cuvette with the blue round top → Electroporation
- room 434: the electroporator for the C2C12 cells → 130 V → pulse length: 30 ms → number of pulse: 5
1) empty cuvette with PBS → start (for cleaning) 2) pulse the cells
- put the cells on ice - add in each cuvette 1 ml HS (HS for the C2C12 cells)
- in every 6 cm dish: 3 ml HS