磷酸钙转染法制备慢病毒的protocol
Production of lentivirus by Calcium phosphate in 293T cells
This protocol was adapted for 10 cm dish.
Day 1: HEK293T plating
Plate 2.5~3×106cells/10 cm dish in DMEM+10?S(+1% L- Glutamine) Day 2: Transfection
There will be around 70% confluence.
Two hours before transfection, replace the medium with 10 ml fresh medium preheated at 37℃. For each 10 cm dish, prepare the following transfection mix: 10.0 ug GFP(or other plasmd of interest) 10.5 ug pLP1 10.5 ug pLP2 9.0 ug pVSVG. Then add in this order:
293.3 ul of TE buffer (0.1×, pH 8.8) 155.6 ul d.d.water
50.2 ul CaCl2(2.5M)Briefly mix, then add 500 ul 2×HBS, dropwise under agitation by vortexing at least 1~2min.
Wait at least 5min(no more than 30min) at RT.
Add dropwise 1ml/10cm dish of the precipitate, and mix gently by rotating the plate.
Day 3: Observe the cells and change the media
Observe the cells. They should be reaching confluency. If a visible marker (such as GFP) is present in the lentivector plasmid, transfection efficiency may be assessed visually. Ideally, transfection efficiency should be >90%.Remove medium around14~16h post-transfection and put ≈ 8ml/dish of fresh pre-heated medium.
Day 4: Collect first harvest of supernatant
Collect and pool supernatant (8 ml/dish) from dishes at 48 h post-transfection and store at -80℃. Add 8ml of fresh DMEM +10% FBS +1% L-Glutamine to each dish. Incubate dishes at 5% CO2, 37 °C overnight.
Day 5: Collect second harvest of supernatant
Collect supernatant from dishes (8ml/dish) at 72 h post- transfection. Pool supernatant from first and second harvests. Centrifuge at 3000 × g for 15 minutes at 4℃ to remove cells and cell debris.
