I am using 2.5 ug DNA:3 uL of Lipofectamine 2000.
I am using OMEM (serum-depleted media) for the transfection.
I have tried transfecting both adherent cells and cells in culture.
I am plating the density in a six well plate so that the cells are only about 30-40% confluent after transfection.
But I am currently only getting about 50% (via GFP), and this is not enough to see depletion.
If you are using 6well plate you should use DNA (4ug) and 10uL Lipo2000. The confluence on the day of transfection should be around 60-80%.
- How long are you leaving your cells with lipo/DNA complex?
- Are changing media some time later?
- Adherent cells are easier to transfect
- How long after transfection are you analyzing GFP expression?
the ratio is obviously wrong. after transfection for a while, you should add serum to improve cell growth and health. even you can transfect in serum containing medium, check your protocol.
Your protocol using 2.5 ug DNA:3 uL of Lipofectamine 2000 is not following the manufacture's datasheet.
It recommends using at least 2x lipofectamine.
Moreover, plating cell in too low density might not increase transfection efficiency.
So, I recommend you to plate cell at least 50% confluence and increase amount of lipofectamine used, 2 or 3 fold might work.
PS. I have used lipofectamine 2000 for 5 years, I don't know that this is only my imaginary or not, but I feel like that the lipofectamine2000 is less effective than the older lot. :(
Andre --
- I have tried leaving the DNA:lipofectamine for a short course (6 hrs) and a long course (24 hours)
- I do change the media at 24 hours, replacing the OMEM/DMEM mix with DMEM containing 10% serum
- I start analyzing GFP expression at 24 hours using a fluorescent micosocpe
Attila --
Thank you immensely. I am mixing the DNA/lipid complexes in OMEM, but then I am adding that to the growth media that I plated the cells in and that contains 10% FBS. I'll try 2 ug and 3 ug of DNA with double or triple as much lipofectamine as a titration.
Wareerat --
Do you think that at 50% confluence with fast dividing cells like HEK293T, the protein expression in non-transfected cells will still cover up the depletion from transfected cells?
I always used 293T to produce lentivirus and for Confocal microscopy. I would transfect at 60% confluence and analyze 48h later.
In my opinion, there are two problems. The first one is transfection efficiency and the second is knockdown condition.
I assume that you cannot detect your protein being knockdown and you think transfection efficiency was the reason.
May I ask? when did you detect your protein target?
Was it the same day as GFP detection? 24 hrs?
By the way, if you use shRNA, establishing knockdown stable cell lines might be the better option.
If you are using a plasmid to produce your ShRNA, the most efficient and less toxic method for HEK293T cells is still calcium precipitation. You should transfect the cells at 50% confluence.
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