Protocol: Titering Concentrated Lentivirus (click to download .docx)
Adam Skibinski/Kuperwasser Lab, created 10-27-10
Reagents Needed:
-Concentrated lentivirus
-6 well adherent plate
-Protamine sulfate 1000x (-20 degree TC freezer in hallway)
–Drugs for selection (e.g. blasticidin or puromycin, -20 degree freezer in TC room)
-10% formalin
–Crystal Violet stain
Day 1
- Plate out 293T cells in a 6-well plate at 500k cells/well (2ml 293C media per well)
- Add concentrated virus to the wells in amounts of 10µl, 1µl, or 0.1 µl. Use dilutions of the virus in media or PBS to add the 1µl and 0.1µl amounts. Add protamine sulfate to the wells at a dilution of 1:1000.
Day 2
Day 3+
Formula for calculating titer:
Titer (infectious particles/ml) = colonies/doubling time (4 for 293T) * dilution factor (e.g. 10,000 for 0.1 µl of virus added, 1,000 for 1µl added, etc.)
