Lentivirus Production
Reagents:
- Mammalian cell transfection kit from Chemicon, #S-001.
- 0.1 X TE, I usually dilute the elution buffer from Qiagen Minoprep kit with the clean H2O from mammalian cell transfection kit. Filter before use.
- IMDM medium from Invitrogen #12440-079.
- 10% FCS prepared by adding 110 ml FCS, 11 ml Strep, to 1000 ml IMDM.
- Syringe driven filter unit of 0.22 um from Millipore, #SLGV033RS.
- Note: Use early passage of 293T cells which give better yield of virus. High efficiency of transfection is the key to get high titers of virus. With the transfection kit, I usually get more than 85% transfection efficiency.
1. 24 h before transfection, plate 4.5 X 10E6 cells in 10 cm plate.
Note: I usually do not count cells. Instead, I split one 10 cm plate cells of 80-90% to 2 plates 24 h before transfection.
2. 2h before transfection, change medium to fresh IMDM of 9 ml.
3. For each plate, make
complex I
VSVG: 3 ug Rev: 2.5 ug pMDL: 5 ug 305: 12 ug (for 305 with insert of 1kb, use 15 ug. For 277 and 277-ShRNA, use 10 ug) 0.1 X TE: Fill to 430 ul Add 60 ul CaCl2 solution. Then incubate at RT for 5 min.
Complex II
H2O: 440 ul 10X HBS: 50 ul NaOH: 7.5 ul Phosphate: 5 ul
4. Combine I and II by bubbling DNA (complex I) into complex II.
5. Incubate at RT for 30 min in the hood. (Do not turn on the UV!)
6. Pipette over cells while swirling the plate.
7. Change medium after 16 hours to 8 ml IMDM.
8. Collect virus after 24 hours, and store the virus at 4C wrapped by aluminum foil. Refill the plates with fresh 8 ml IMDM.
9. Collect for another 24 hours.
10. Alternatively, fill the plate with 10 ml fresh IMDM at step 8. Collect the supernatant after 30 hrs.
11. Freeze down the virus at -80C with 1 ml aliquot.
Based on protocol from Follenzi A with modifications.