Retrovirus production in Plat-E cell

Retrovirus production in Plat-E cell

Material

Plat-E cell culture

  1. Culture by DMEM/10% FBS/PS(+) on collagen-coated 10 cm dish.
  2. 1x10^6 cells/dish will be confluent after 2 days.

Day 1

  1. Change medium to 10 ml DMEM/10% FBS/PS(-).

Day 2 (confluent)

  1. Change medium to 5 ml DMEM/10% FBS/PS(-).
  2. Add 20 ug retroviral vector to 1 ml Opti-MEM in 1.5 ml tube.
  3. Tap to mix.
  4. Add 30 ul TransIT-2020 to mixture.
  5. Tap to mix.
  6. Incubate at RT for 15 min.
  7. Drop mixture onto dish, not mix.
  8. Culture o/n.

Day 3

  1. Change medium to 5-10 ml DMEM/10% FBS/PS(-).

Day 4

  1. Harvest supernatant as retrovirus sol.
  2. Filtrate by 0.45 um filter.
  3. Aliquot and store at -80C.