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This is intended as a guide only; for full experimental details please read the references provided.
Protocol for transient gene expression in adherent HEK293 cells using PEI STAR™ transfection reagent .
We list recommended reagent quantities per 106 cells in the table below. If performing many very similar transfections (e.g. making many similar viruses), we would recommend co-varying on these parameters over optimization ranges ﬁrst.
|PEI STAR™ Concentration (per 106 cells)||3.0 μg||2.0 to 4.0 μg|
|DNA Concentration (per 106 cells)||1.5 μg||1.0 to 2.0 μg|
|PEI STAR™/DNA Complex Time||10.0 min||5.0 to 15.0 min|
Even if performing co-transfection, keep the total DNA concentration range the same (1.0 to 2.0 μg/106 cells).
If high toxicity is observed, then reduce the quantity of PEI and/or DNA ﬁrst. We do not recommend replacing the media post-transfection to reduce toxicity except as a last resort.
Seed the cells the day before transfection to reach 50-80% conﬂuence on the day of transfection. Seed approximately 50,000 cells per cm2
1. Measure the cell density to determine the transfection parameters based on the number of viable cells.
2. Dilute 1.0 μg pDNA per 106 cells in 5% ﬁnal culture volume of DMEM or media. Mix well.
3. Dilute 3.0 μg PEI STAR™ per 106 cells in 5% ﬁnal culture volume of DMEM or media. Mix well.
4. Mix the solution of pDNA and PEI STAR™ together by quickly and brieﬂy vortexing or inverting the tube several times.
5. Allow the mixture of pDNA and PEI STAR™ to rest for 10 minutes at room temperature.
6. Gradually add the pDNA and PEI STAR™ mixture to the cells dropwise while swirling the plate.
7. Incubate the cells at typical incubation conditions.
Boussif et al (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc.Natl.Acad.Sci.U.S.A. 92 7297. PMID: 7638184.
Longo et al (2013) Transient mammalian cell transfection with polyethylenimine (PEI). Methods Enzymol. 529 227. PMID: 24011049.
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