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Transfection of HEK 293 Cells
Calciumphosphat Coprecipitation Method
Solutions:
| for 200 ml | ||
| 2XCaCl2: | 500 mM CaCl | 14.7 g |
| 1 mM Tris, pH7.5 | 0.2 ml of 1M stock | |
| 0.1 mM EDTA | 40 ul of 0.5 M stock | |
| pH 7-8, store at 4°C |
| for 200 ml | ||
| 2XHBS: | 50 mM Hepes | 2.383 g |
| 280 mMNaCl | 3.273 g | |
| 1.5 mMNa2HPO4 | (0.0534 g) | |
| pH 7.1, store at -20°C in aliquots |
Transfection (TF):
TF - 8 hrs:
Prepare 9-12 dishes of 6 cm diameter with 4ml culture medium.
Trypsinize cells from one confluent flask , add 10 ml medium and distribute the cell solution into the dishes (1ml each).
TF:
Equilibrate the transfection reagents (2XCaCl2- and 2XHBS) at room temperature. Prepare10 µg of DNA in 130µl H2O in an Eppendorf. Add 130µl of 2XCaCl2-Solution.
While vortexing, slowly add this solution (260 µl) to a 15 ml Falcon
tube containing 260 µl of 2XHBS-Solution. You will see the formation of
a weak precipitate. Incubate for at least 45 minutes at RT. Mix once
more and slowly (dropwise) add the precipitate to the cell dish, trying
to cover the whole surface.
TF + 16 hrs:
Medium exchange: Aspirate the medium from the cells and add 5 ml of fresh medium.
TF + 48-72 hrs:
Harvest the medium and start assay (ELISA or Western Blot)
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