Lentivirus protocol

  1. Split 293T cells the day prior so that they are 80-90% confluent on day of transfection.
  2. Change media on cells just prior to transfection. Use 10% BGS DMEM supplemented with pen/strep(1:100) and l-glutamine(1:100). 10-15mL media per 10cm plate.
  3. Follow TransIT-293 directions for transfection of lenti and helper plasmids. For a 10cm plate, use 9ug lenti plasmid and 2ug each helper plasmid for a final 15ugs of DNA. Helper plasmids are pRRE, VSVG, and RSV-Rev. Alternately, use calcium phosphate transfection (see Porteus Lab lentivirus protocol).
  4. Harvest viral supernatant on day 2 post transfection. Clear debris/cells by spinning down at 1500rpm for 10 minutes at 4C. Remove supernatant to a new tube.
  5. If concentrating the virus, follow Lenti-X instructions.
  6. Aliquot virus into eppendorf tubes, label and store at -80C.
  7. To infect cells with lentivirus, thaw tubes on ice.