I have been trying to transfect HEK293 cells with Transfast and Lipofectamine and have not been able to confirm successful transfection. Does anyone have advice or I good protocol I could use?
Have you tried GenePORTER 3000? It's a same-day cell plating/transfection protocol with >90% efficiency. Simply detach healthy log phase cells, count and plate at high density and transfect with GP3K an your plasmid of choice.
Go to: http://genlantis.com/ and click on our banner ad for GP3K. That will take you to the data and manual.
I use lipofectamine 2000. The same-day transfection and plating or reverse transfection will also help boost your percent transfected. Good luck!
We use FuGene-6 with HEK293 cells pretty routinely and get good results. We just use the standard protocol, but this reagent can be a little pricey.
Optimizing transfections can be so much fun....
You should be able to get 80% transfection efficiencies with home made solutions for calcium phosphate transfections. The quality of the cells is probably the most important factor. They should never be allowed to become over-confluent, so split 1:10 every 3 days.
As well as suggesting an 'Ultra' version of lipofectamine such as Lipofectamine LTX or plus, you could check that your detection method for detecting an efficient transfection is working properly. I'm not sure what your endpoint is for the successful transfection e.g. which protein you are trying to overexpress, but you could perhaps try a few different transfection reagents and insert GFP or something else for the readout.
In my experience Lipofectamine works like a charm in many cells, including HEK293. I agree with parvoman that one of the main considerations is how well the cells are growing. If you let them reach confluency, even if this happened a few passages ago, the transfection efficiency will be significantly diminished. If this is the case, you may simply want to thaw a new tube and start your cells again because you will likely come across the same problem with other transfection reagents.
Transfection protocols for HEK293 cells are available here:
i have a question regarding confluency of HEK293 cells for transfection.Yous uggested to thaw a new vial of cells if the cells had reached confluency( even if it was few passages before). Does it mean that once HEK293 cells reached 100% confluency, it CANNOT be used at all for transfection expts?
It might not feasible to find a 293T cell line in a lab that might not have become 100% confluent at least once during the passages.
You can definitely transfect cells that have reached 100% confluency, that is perfectly fine. My point was that if you use cells that have reached 100% confluency your transfection efficiency will not be as good as when you use cells that have not reached 100% confluency.
I also heard about the be cautious not to let 293 ever get confluent statement from others. Do you ever come across any reference on that issue.?Or probably more importantly, what if the cell lines stored in our liquid nitrogen had once became confluent. What should I do with it? Would it help if I thaw a vial and grow then many passages without letting them confluent. Would this reverse their transfectability again? Or how would you suggest me to do? Thank you.
I agree with a previous reply about using the Lipofectamine LTX, It has worked great for me. This may seem ridiculously trivial but, do you add FBS to your transfection media? I made the mistake on accident, and it completely failed, so use an FBS free media (I use opti-MEM), then post-transfection you can switch to FBS supplemented. Regarding ability to confirm transfection, what plasmid are you using?