Hi Kajal,
I've transfected HEK293T cells with Lipofectamine LTX + Plus frequently without any trouble. You can achieve high efficiency transfection (as measured with a GFP expression vector) by following the conditions outlined in this Thermofisher document:
https://www.thermofisher.com/uk/en/home/references/protocols/cell-culture/transfection-protocol/transfecting-plasmid-dna-into-hek-293-cells-using-lipofectamine-ltx-reagent.html
For a 6-well plate, I've been using the following conditions which work well in my hands and broadly hit >80% efficiency with little apparently toxicity:
- Seeding density: 6.25x10^5 cells per well (seeded 24 hrs previously)
- Endotoxin free plasmid DNA: 2500ng
- Lipofectamine LTX - 12.5uL per well
- Plus Reagent: 2.5uL per well (1uL per 1000ng DNA)
- Complex time: 30 minutes
Best wishes,
Connor
@ Connor E Woolley Thanks a lot for your help. I will definitely go through it.
If I were you I would use PEI as your transfection reagent.
http://www.polysciences.com/default/polyethylenimine-linear-mw25000
It is extremely inexpensive when compared to commercial reagents. In fact you can prepare 100s of ml of reagent (and store it) for the price it costs for other reagents. We use it routinely and get >95% transfection efficiency in HEK293 cells.
Our protocol is as follows.
Seed cells in 10cm dishes at 4.5E6 cells/dish in 10ml medium (the day before)
Prepare complexes (7ug/DNA, 21ul 1mg/ml PEI) in 1ml serum free medium
Complex at RT for 30 mins
Add dropwise to cells.
I hope this is useful.
Gary James Spencer Thanks a lot for your suggestion. I am using PEI only but due to some experimental requirement I want to shift on this. Well, I will try this ratio of DNA and PEI.
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