I would like to transfect 293T cells with Lipofectamine 3000. This is the first time I will have used Lipofectamine 3000, previously I only used Lipofectamine 2000, and I am wondering if anyone has found an optimal Lipo 3000 / DNA ratio for this cell line, particularly for packaging lentivirus. I of course realize I may have to make multiple attempts to find the optimal ratio for my particular application but I would love some advice on where to start.
Lately, I tried using lipofectamine 3000 instead of 2000. Previously I used 1:3 ratio of DNA:Lipo2000, but I thought we needed less amount of lipo3000 than 2000. So I used 1:2 DNA:LIpo3000 and it worked. I did not try to optimize my protocol, but from what I heard and saw you should use less amount of lipo 3000 than 2000
We successfully packaged Lentivirus by using Lipofectamine 3000 in 293T cells. The optimum conditions for packaging Lentivirus in 6-well plate are:
The day before transfection:
Seed 293T cells in 6-well plate at 1.5x10^6 cells/well;
The day transfection (Per DNA-Lipo P3000/Lipo 3000 Reagent complex):
3ug Packaging DNA + 6ul of Lipofectamine P3000 (DNA: P3000 = 1ug : 2ul);
7ul of Lipofectamine Reagent;
The total complex volume is 300ul --- 150ul(DNA-Lipo P3000) + 150ul(Lipo 3000 Reagent).
You can scale up and down the cell seeding density, the amounts of DNA, reagents and complex volume for your transfection by comparing the growth area of your plate format with the 6-well plate. The growth area ratio will be your transfection scale up and down ratio.
Hi Stephan, you should also consider an alternative to your current reagent such as DreamFect Gold at a ratio of 2/1 or 3/1 Best Regards Olivier
293T cells are very easy to be transfected and lipo 3,000 is much easily use than lipo 2,000. I usually follow the protocol they provided and have good transfection efficiency.
Good luck.
Does any one use lipo3000 to transfect GP2-293? What's protocol is that?
Thanks!
HEK293T cells can be very efficiently transfected by polybrene, which costs nothing compared to Lipofectamine 2000 or 3000.
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