I am using lipofectamine to transfect plasmids to produce lentiviruses in 293T cells. The vectors I am using belong to the second generation of lentivectors, pMD2.G (envelope plasmid) and psPAX2 (packaging plasmid) + pLenti6/V5-D-TOPO modified to be 9kb long. the protocol I was told to use is>
OMEN + plasmids to a final volume of 500 uL
OMEN + lipofect. 16ul to a final vol. of 500 uL
incubate 2 min + mix + vortex
incubate 15 min
aspirate old mediom frim 10 cm dish and and 3ml of fresh medium
+add solution to dish
change medium after 5+7 hours
Anyway I do not know which amunt of plasmids I should use. From a previous trial with calcium phosphate transfection I used 22.5 ug of clonned construct, 7.9 ug envelope plasmid and 14.6 packaging plamid but I am not sure if it will be correct for this method.
Thanks! and do you know if 0.22 um filtering could be used or instead better 0.45 um?
Here is the protocol I use, in brief.
1. Transfect HEK293T (T75, 90% confluent) cells with 21ug of total DNA (3 DNA constructs) using Lipofectamine LTX (105uL) and Plus Reagent (21uL). The DNA/lipid complexes are prepared in 4.2mL of OptiMEM.
2. Add dropwise onto cells in a T75 with 16mL DMEM.
3. Incubate for 24hrs (typically achieve >80% transfection efficiency). Change medium to DMEM or target cell medium. Add only 10mL.
4. Incubate a second 24hrs. Collect supernatant containing LV and add 10mL medium to HEK293T cells.
5. Spin supernatant at 2000rpm for 10 min. Transfer supernatant to a new tube, avoiding pellet.
6. Add polybrene (8ug/mL) to supernatant and mix gently. Add supernatant to target cells. Incubate for 48 hrs.
Result of a recent Lentivirus transduction experiment is shown below. I often need to dilute the supernatant by 1/4 to avoid negative impact on cell viability, but still achieve about 90% transduction efficiency. The target cells are human primary corneal endothelial cells. Best of luck.
- Badges
- Science topic