Electroporation of C2C12 cells

material

Transfection

• 1x Steinberger Medium
• 1x DMEM without serum

- 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