Hi, I am facing some problem with my transfection experiments. I am trying to transfect a pcDNA expression vector into HEK293 cells with Lipofectamin LTX and Plus reagent from Invitrogen. I started the transfection following manufacturer’s recommended protocol, but could not detect the protein. If anybody has a transfection protocol optimized for HEK293, please share with me. That will be a great help.
HEK are easy-to-transfect cells, the standard protocol should work. What are your conditions? Is your plasmid too big? Do you have a control? Please, let us know some more info to help you.
Hi there,
I use polyethyleneimine (PEI) for transfecting HEK 293.
The best PEI as transfection agent is the 25kDa linear from Polysciences.
I first make 150 mM solution of PEI in water and sterile filter and store at -20C till use.
For making transfection mixture, I add 1ug of DNA (diluted in 20 ul of 5% glucose) to 0.12 ul of PEI (diluted in 20 ul of glucose) and incubate for 20 min at RT before adding directly to cell culture.
1-2 ug plasmid/ml culture gives best result.
At the time of transfection cells should be around 2.5X10e5 cells/ml.
Better if cells are not in clumps and actively dividing.
There is no need for serum free medium and subsequent media change, as required in most lipid based transfection.
After 72h of transfection you may see the results.
Best
Sudeep
Sure! Here's our protocol for our HEK293-optimized transfection kit (https://altogen.com/product/hek-293-transfection-reagent-epithelial-kidney-cells/):
1. Plate 10,000 - 15,000 HEK293 cells per well in 0.5 ml of complete growth medium 12–24 hours prior to transfection
2. Wash with 1xPBS and add 0.5 ml of fresh growth medium
3. Prepare transfection complexes by mixing 40 µl of serumfree medium, 4.5 µl of transfection reagent, and 500 ng DNA (or mRNA) or 30 nM - 50 nM of siRNA (or microRNA)
4. Incubate transfection complexes at RT for 15 - 30 minutes
5. Optional: Add 2 µl of Complex Condenser. This reagent reduces the size of transfection complex, therefore increasing transfection efficiency; however it may increase cell toxicity
6. Add prepared transfection complexes to 0.5 ml of complete growth medium with HEK293 cells (from step 2)
7. Incubate cells at 37ºC in a humidified CO2 incubator 8. Assay for phenotype or target gene expression 48 - 72 hours after transfection
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