- "Spinoculation" transfection expriment
"Spinoculation" transfection expriment
- Try centrifuge spin approach to increase transfection efficiency
- See notes from
- Use U2OS cells
- KAH126/MV2, spin
- mock, spin
- KAH126/MV2 conventional
- mock conventional
- Set up Lipo-DNA and mock complexes for 6-well plates (standard Lipo LTX protocol): 2000 μg/ 20 μL DNA, 570 μL Opti-MEM, 2.5 μL PLUS, 7.5 μL Lipofectamine LTX
- Harvest U2OS cells from T-75 flask (~1.0E6 cells/ mL)
- Transfer ~5.0E5 cells into sterile 15 mL conicals. Fill up to final vol. of 3.4 mL with growth medium
- Add 0.6 mL Lipo-DNA or mock complexes. Swirl to mix.
- Spin at 3000 rpm (800 - 1000 xg) at room temp for 25 min. Note: centrifuge is IEC Centra-CL2 with rotor 215.
- Transfer most of supernatant into 6-well plate wells
- Flick to disrupt the pelleted cells. Use 1 mL of growth medium to transfer cells to wells.
- Remove 0.5 mL growth medium from each well
- Drop-wise, add Lipo-DNA or mock complexes to wells
Grow all cells overnight at 37°C/5% CO2. Check for RFP expression tomorrow