Lipofection into myoblast for luciferase assay
Material
- Myoblast
- Growth medium for myoblast (Ham's/F10, 20% FBS, 0.2 ug/ml bFGF, PS)
- TransIT-2020 (Mirus MIR5400, -20C)
- Opti-MEM (Invitrogen 51985, 4C)
Day 1
- Seed 5x10^4 myoblasts/well with 200 ul growth medium on collagen-coated 48-well plate.
Day 2
Volumes described below are for 1 well.
- Add 1 ug DNA to 100 ul Opti-MEM in 1.5 ml tube.
- Tap to mix.
- Add 1 ul TransIT-2020 to mixture.
- Tap to mix.
- Incubate at RT for 15 min.
- Drop mixture onto well, not mix.
- Culture for 6 h.
- Change medium to 200 ul growth medium.
Day 3 (after 24 h of lipofection)
- Check myoblast.
Day 4 (after 48 h of lipofection)
- Wash by 200 ul PBS once.
- OPTIONAL: Save plate at -80C.
- Add 65 ul PLB.
- Scrape well by blue tip to harvest cell lysate.
- OPTIONAL: Save lysate at -80C.
- Centrifuge at 13000 rpm at RT for 3 min.
- Use supernatant to measure Luc activity.