Transfection of HEK293T cells: Difference between revisions
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Created page with "{| class="wikitable" !Materials ! |- | |Plasmid DNA |- | |2x HBS- buffer, pH 7.05 (correct pH is extremely critical !!) |- | |2.5 M CaCl2 (sterile) |- | |25 mM chloroquin (sterile) |} '''Procedure''' * 1 day before transfection: cells split 1:3 up to 1:5 in 10cm dishes * Next day: Set up for each 10cm dish: 500 μl ddH2O (sterile) in 14 ml PP Greiner tube * Add 5 μg (or adapted amounts) of plasmid DNA * Add 500 μl 2x HBS buffer, mix sample * Add drop-wise and duri..." |
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|25 mM chloroquin (sterile) | |25 mM chloroquin (sterile) | ||
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'''Procedure''' | '''Procedure:''' | ||
* 1 day before transfection: cells split 1:3 up to 1:5 in 10cm dishes | * 1 day before transfection: cells split 1:3 up to 1:5 in 10cm dishes | ||
Latest revision as of 16:33, 15 February 2025
| Materials | |
|---|---|
| Plasmid DNA | |
| 2x HBS- buffer, pH 7.05 (correct pH is extremely critical !!) | |
| 2.5 M CaCl2 (sterile) | |
| 25 mM chloroquin (sterile) |
Procedure:
- 1 day before transfection: cells split 1:3 up to 1:5 in 10cm dishes
- Next day: Set up for each 10cm dish: 500 μl ddH2O (sterile) in 14 ml PP Greiner tube
- Add 5 μg (or adapted amounts) of plasmid DNA
- Add 500 μl 2x HBS buffer, mix sample
- Add drop-wise and during constant mixing on vortex 50 μl sterile CaCl2 to sample
- Incubate for 5 min at room temperature to allow DNA/Ca2+ precipitate formation
- During DNA/Ca2+ precipitate formation, add 10 μl of chloroquin to cells in 10 cm dish
- Add drop-wise 1 ml of DNA/Ca2+ precipitate sample on cells in 10 cm dish (“carpet bombing”), be carefull not to detach cells
- Incubate cells for 6- 8 h in incubator
- Remove medium and add 8 ml/dish of fresh medium carefully onto cells
- Expression of transgenes is usually highest on day 2 after the transfection