STEPS:
- One day before transfecting, seed cells (in a well plate) so that they are 70-90% confluent at time of transfection.
- Required Materials: Eppendorf tubes, Opti-MEM Medium, PLUS Reagent, Lipofectamine LTX Reagent.
- Dilute Lipofectamine in Opti-MEM.
- Dilute DNA in Opti-MEM, then add PLUS.
- Add diluted DNA to diluted Lipofectamine in a 1:1 ratio, to form DNA-lipid complex.
- Incubate at room temperature for 5 minutes.
- Add DNA-lipid complex to cells (wells).
- Incubate cells for 1-3 days at 37C.
- Visualize/analyze transfected cells (flow cytometry).
n-WELL PLATE PROPORTIONS:
| STEP | COMPONENT | 6-well | 24-well | 96-well |
|---|---|---|---|---|
| 1. | Adherent Cells | 0.25-1 x 10^6 | 0.5-2 x 10^5 | 1-4 x 10^4 |
| 3. | Opti-MEM Medium | 150uL x 4 | 50uL | 25uL x 4 |
| Lipofectamine LTX Reagent | 6, 9, 12, 15 uL | 2uL | 1, 1.5, 2, 2.5 uL | |
| 4. | Opti-MEM Medium | 700uL | 250uL | 125uL |
| DNA (0.5-5 ug/uL) | 14ug | 10ug (total) | 2.5ug | |
| PLUS Reagent | 14uL | 5uL | 2.5uL | |
| 5. | Diluted DNA (with PLUS) | 150uL | 52uL | 25uL |
| Diluted Lipofectamine LTX | 150uL | 52uL | 25uL | |
| 7. | DNA-lipid complex (per well) | 250uL | 50uL | 10uL |
| FINAL DNA (PER WELL)** | 2500ng | 1000ng | 100ng |
NOTES:
** For co-transfection, a TOTAL of xng of DNA should be added to each well: if 2 plasmids, add (x/2)ng of each, and so on.
In blue: To be tested (optimal volume has not yet been determined).
