Transfecting C2C12 Cells
From SDMRC
Transfecting C2C12 Cells
- siRNA against Cullin 3
- siRNA with other sequence
Materials
- Two tubes (1.7ml) per well (12 tubes total)
- 6 for control
- 6 for treatment
- Label tubes
- Control- tube #1, tube #2
- Cullin 3- tube #1, tube #2
- In tube #1: add 190 ul of DMEM in each
- In tube #2: add 196 ul of DMEM in each
Method
- Add 10 ul of pre aliquoted 5 mmol siRNA. Centrifuge, flush tube to mix
- Add 4 ul of transfection reagent (blue cap) into tube #2 Centrifuge, flush tube to mix
- *you should have 200 ul total of each solution*
- Incubate at room temperature for 5 minutes
- In the meantime prepare 15 ml tubes (1 tube per well)
- Add 1.6 ml of differentiation media without antibiotic (penicillin/streptomycin)
- 200 ml of DMEM + 4 ml horse serum
- In the 1.6 ml of media add 400 ul of the transfection mix (two tubes each with 200 ul)
- This brings the total volume to 2 ml of solution which is needed for each well to grow
- Place tube #1 (200ul) into corresponding tube #2, flush to mix, change tips between control and treatment
- Set tubes at room temperature for 20 minutes
- Place the 400 ul in each matching tube, flush, and cap
- Add the new media in the wells (change tips in between) and incubate for 24 hours [do one treatment at a time]
- Label which wells are control and treatment
- After 24 hours replace media with normal differentiation media
After the 24 hours
- Wash each well 3 times with 2 ml of PBS and remove
- Add 4% PFA (kept in door) 1 ml per well for 10 minutes, then remove
- Wash 3 times again with PBS, on 3rd wash leave PBS in dish
- Plates can be used in next step or stored in the -20 fridge wrapped in parafilm
