Production of lentiviral particles

Material
	293T cells
	2x HBS-buffer
	2.5 M CaCl2
	25 mM Chloroquin
	Lentiviral vectors

Procedure

1. A confluent plate of 293T cells is splitted 1:5 one day before transfection
2. The transfection reaction contains: 500 µl ddH₂O, 6.5 µg pLKO.1 (with gene or shRNA of interest), 3.5 µg pMD2.G (plasmid #1256) and 5 µg psPAX2 (plasmid #1257)
3. Add 500 µl 2x HBS-buffer
4. While vortexing the reaction, 50 µl of 2.5 M CaCl₂ are added dropwise
5. Incubate for 10 min at room temperature
6. During the formation of Ca2+/DNA precipitates, add 10 µl of chloroquin (25 mM) to each 10 cm culture dish with cells growing in a volume of 10 ml medium
7. Distribute the ~1ml of transfection mix dropwise onto the cell culture plate (carpet bombing)
8. Aspirate chloroquin-containing medium after 6 h and replace with 6 ml fresh DMEM / 10% CS / Pen/Strep
9. After 48-72 h, the virus-containing supernatant is collected and centrifuged for 7 min at 2000 rpm and 4°C
10. After 48-72 h the virus-containing supernatant is collected and centrifuged for 7 min at 2000 rpm and 4°C
11. Sterile filtration of supernatant (0.2 µm pore size) and directly employ in transduction of target cells or aliquote and quick-freeze the supernatant in liquid nitrogen, storage at -80° C