I recently tested PEI for production of GFP lentiviruses in HEK-293T cells. Important parameters in my protocol were as follows:
- PEI solution (1 ug/uL): branched PEI (sigma408727) was diluted 1:1000 in water -> pH was adjusted to 6 -> filter-sterilization
- Transfection: 9 uL packaging plasmids mix (invitrogen) + 3 ug pLenti6.3-GFP + 1mL serum free DMEM + 36 ul PEI solution -> vortex (5 times, 1s each) -> 15 min @ RT -> added to cells dropwise (~5*10^6 cell/10 cm plate)
I checked the packaging cells for GFP expression. The lipofectamine control (with manufacturer protocol) was fine but PEI didn't work at all. What may be the problem with my protocol?
Thank you Arjan.
Actually I vortexed the tube 5 times and each time for 1 second.
I will check your protocol but I just wonder how much serum free medium do you use for dilution of DNA and PEI? Are the cells incubated in low-serum medium during the transfection?
And yes I'm sure about the GFP plasmid!
Many thanks again, I really need that good luck!
I use linear PEI(Polysciences, Cat# 23966), PEI solution concentration is 7.5mM, PH is 8 and use N/P ratio 40. For a 150mm dish, I use total DNA 10.5ug. The transfection efficiency is >90%. I use 10% FBS+1%PS in DMEM. Good Luck!
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