I'm trying to transfect a large plasmid (about 15 kb) into HeLa cells using Lipofectamine 2000. I've tried following the manufacturer's recommendations but can't seem to get much, if any, transfection at all. I do the transfection alongside the empty vector (about 6 kb) and I get efficient expression of that after checking via WB so I don't think any of my reagents are in question.
I'd appreciate any troubleshooting recommendations from anyone or a whole other method of transfection of large plasmids if people have had better experience with them. Thanks in advance!
I had a similar problem, so I doubled the quantity of plasmid transfected and I got a good result ( I performed wb and luc assay).
Maybe you could try to gradually increase the quantity of the material transfected to optimize.
Hello try the transfection with the GFP plasmid.
see attached info for more info.
Good luck!
I know some people use electroporation for large vectors (but I have never used it myself in human cells, I electroporate up to 12Kb vectors into Dictyostelium cells easily, but I'm not sure my protocol will work for human cells, check the answer posted in this older post https://www.researchgate.net/post/Methods-for-large-plasmid-stable-transfection). For human cells, I have used recently lipofectamine 3000 (together with the 3000 reagent) with better results than the 2000. I got it as a free sample from Thermofisher, maybe you can also ask for one if you want to test it. Also, try to be sure your DNA is very clean, since impurities may decrease your efficiency (which may be more evident using the very large vector).
I agree with Eunice Domínguez-Martín's suggestion. You could try Lipofectamine 3000 reagent, which is better than Lipofectamine 2000. Electroporation is another option, but I remember that the condition should be optimized according to different cells. if you get the correct condition of electroporation, the efficiency will be extremely good.
- Badges
- Science method