Lipofection into 293T cell for luciferase assay
Material
- 293T cell
- DMEM (DMEM/10% FBS/PS(-))
- TransIT-2020 (Mirus MIR5400, -20C)
- Opti-MEM (Invitrogen 51985, 4C)
Day 1
- Seed 5x10^4 293T cells/well with 200 ul DMEM 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 o/n.
Day 3 (after 24 h of lipofection)
- Change DMEM.
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.