Transient Transfection of 293T Cells for Lentiviral Production (PDF)
From Laurie Ailles, Weissman Lab (this is the protocol the Galli Lab uses)
- Seed and incubate 3 x 106 293T cells per 10 cm dish approximately 24 hours prior to transfection, in IMDM with 10% FBS.
- 2 hours prior to transfection, change media.
- Prepare plasmid DNA in 50 ml conical tubes as follows:
Per 10 cm dish: 10 ug transfer vector plasmid
6.5 ug CMVΔR8.74 plasmid
3.5 ug VSV.G plasmid
Bring up to a total volume of 450 ul with diluted TE(If you are transfecting more than one dish, you can mix the appropriate amount of DNA and TE in a single tube, then aliquot it into individual tubes prior to adding the CaCl2) Add 50 ul 2.5M CaCl2 Add 500 ul 2X HBS dropwise, while vortexing at full speed (about 2 drops per second). Immediately add total volume to plate of 293T cells and swirl plate to disperse precipitate. Incubate plates at 37°C. - Change media approximately 16 hours later.
- Collect the media 24 hours later, filter through 0.45 um filter, and store at 4°C; add fresh media to plates.
- Collect media again after another 24 hours. Filter.
- Pool media together and ultracentrifuge in SW28 rotor, 19500 rpm, 2hours 20 min.
- Pour off supernatant, resuspend pellet in a small volume of PBS with 0.1% BSA. Agitate on a rocker or rotator at room temp for at least 30 min to completely solubilize vector. Do a quick-spin to spin out insoluble junk, aliquot, and store at –80°C.
| Diluted TE: | Dilute 1X TE 1:15 in sterile water |
| 1X TE: | 0.5 ml 1M Tris.HCl, pH 7.5 0.25 ml 200 mM EDTA in 50 ml total. Filter through 0.22um filter |
| 2.5M CaCl2: | 18.38 g in 50 ml water. Filter through 0.22 um filter. Aliquot and freeze at –20°C |
| 2X HBS: (Note - store at at –20°C. Do not store in fridge for more than 2 wks) |
8.0 g NaCl |
