Retroviral Production using Mirus Transit-293TRetroviral Production using Mirus Transit-293T
Lentiviral Production using Mirus Transit-293T
Reagents:
293T cells
DMEM + 10% FBS
OPTI-MEM
Mirus Transit- 293T (MIR2700)
General Timeline/protocol
Day 1: Seed 293T cells in DMEM+10% FBS (goal: 60% conflue...
Retroviral Production using Mirus Transit-293T
Lentiviral Production using Mirus Transit-293T
Reagents:
293T cells
DMEM + 10% FBS
OPTI-MEM
Mirus Transit- 293T (MIR2700)
General Timeline/protocol
Day 1: Seed 293T cells in DMEM+10% FBS (goal: 60% confluence at Day2). 6~2-2.2x10 (in 4 mL) per 6cm dish 6~4.5-5x10 (in 9mL) per 10cm dish
Day 2: Transfect 293T cells with lentiviral backbone + HDM-tat+HDM-VSV-G+HDM-Hypm2 (Gag) +PRC-CMV-RaII (Pol)
Note: No change in media necessary before transfection
Day 3: Change media (3mL per 6cm dish, 5mL per 10cm dish).
Day 4: Harvest viral supernatant. Store at 4 degrees (temporarily) or aliquot and freeze on dry ice
or at -80C. Add 3mL per 6cm dish or 5mL per 10cm fresh media.
Day 5: Harvest viral sup and combine with Day 4-sup. Filter virus with 0.45 micron filter (low
protein-binding), aliquot, and store at -80 degrees.
Transfection Protocol with Mirus Transit-293T Reagent
Ratio: 3uL Transit : 1ug DNA.
For 6cm dish: 12.6uL MIRUS : 4.2ug DNA total : 500uL OPTI-MEM
For 10cm dish: 30uL MIRUS : 10ug DNA total : 1mL OPTI-MEM
1. Add serum-free media (DMEM or Opti-MEM) to 15mL conical tube. * 0.5mL media per transfection but always make cocktail for more than needed*
2. Add MIRUS reagent to media and mix by finger flicking gently. *12.6uL (6cm) or 30uL (10cm) per transfection*
3. Incubate at room temperature for 10 minutes.
4. Add DNA to sterile microcentrifuge tubes.
* 3.0ug lentiviral vector + 0.3ug each of tat+VSV-G+Gag+Pol = 4.2ug DNA total (6cm) *7.0ug lentiviral vector + 0.75ug each of tat+VSV-G+Gag+Pol = 10ug DNA total (10cm)
5. Add 500uL media/Transit mixture to DNA tube and mix by pipetting.
6. Incubate at room temperature for 20 minutes.
7. Mix gently by pipetting and add to 293T cells dropwise around plate.
8. Mix media back and forth gently. NO SWIRLING.
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