Transfecting C2C12 Cells

Transfecting C2C12 Cells

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

1. Add 10 ul of pre aliquoted 5 mmol siRNA. Centrifuge, flush tube to mix
2. Add 4 ul of transfection reagent (blue cap) into tube #2 Centrifuge, flush tube to mix
3. *you should have 200 ul total of each solution*
4. Incubate at room temperature for 5 minutes
5. In the meantime prepare 15 ml tubes (1 tube per well)
6. Add 1.6 ml of differentiation media without antibiotic (penicillin/streptomycin)
7. 200 ml of DMEM + 4 ml horse serum
8. In the 1.6 ml of media add 400 ul of the transfection mix (two tubes each with 200 ul)
9. This brings the total volume to 2 ml of solution which is needed for each well to grow
10. Place tube #1 (200ul) into corresponding tube #2, flush to mix, change tips between control and treatment
11. Set tubes at room temperature for 20 minutes
12. Place the 400 ul in each matching tube, flush, and cap
13. Add the new media in the wells (change tips in between) and incubate for 24 hours [do one treatment at a time]
14. Label which wells are control and treatment
15. After 24 hours replace media with normal differentiation media

After the 24 hours

1. Wash each well 3 times with 2 ml of PBS and remove
2. Add 4% PFA (kept in door) 1 ml per well for 10 minutes, then remove
3. Wash 3 times again with PBS, on 3rd wash leave PBS in dish
4. Plates can be used in next step or stored in the -20 fridge wrapped in parafilm