Product No. SHP001
Protocol provided by MISSION® Team
The MISSION® Lentiviral Packaging Mix is an optimized formulation of two plasmids expressing the key HIV packaging genes and a heterologous viral envelope gene. MISSION® lentiviral particles are generated from three main components:
0.1 Seed HEK293T cells (20,000 cells/well of 96-well plate) in complete DME media.
1.1 Thaw the vial of Lentiviral Packaging Mix at room temperature and place on ice.
1.2 Put three empty polypropylene tubes on ice.
1.3 Add 1 µL of Lentiviral Packaging Mix per well to the first tube.
1.4 Add 15 µL of serum free DME and 0.6 µL transfection reagent, Fugene 6, per well into the second tube.
1.5 Add 0.1 µg/well of shRNA transfer vector into the third tube.
1.6 Combine all transfection cocktail components together in one polypropylene vial.
1.7 Mix gently by pipetting up and down.
1.8 Incubate at room temperature for 15 min.
1.9 Equally divide the mixture among the well to be transfected.
1.10 Incubate cells at 37 °C overnight in tissue culture incubator.
2.1 16 hours post-transfection, gently remove media from the transfected cells (avoid disturbing cells) and replace with 100 µL of the pre-warmed complete media per well of 96-well plate.
2.2 Incubate cells in incubator (37 °C and 5% CO2) for an additional 24 hours.
3.1 Double glove before proceeding to 3.2.
3.2 Gently remove supernatant, now containing virus, and place it in a collection plate.
3.3 Add fresh complete DME to the wells and incubate cells at 37 °C overnight.
3.4 Cover, and store collection plate at 4 °C.
4.1 Double glove before proceeding to 4.2.
4.2 Remove second viral supernatant from wells and add it to the collection plate from the day before.
4.3 Determine titer by p24 ELISA.
4.4 Store virus tightly sealed at –80 °C.
It was determined experimentally that the first harvest's titer is slightly higher than the second, but pooling the two will not significantly decrease the overall titer of the sample.
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