Dear Members,
Kindly help me if anyone ever had used or having an idea about advanced MEM in cellular transfections. I am facing low transfection efficiency with lipofectamine 3000 when compared with Viafect (yet viafect is not that great too). I am using CFPAC, CAPAN2, PANC 10.5 and PANC1 cell-lines.
Any bright ideas to achieve higher transfection efficiency? kindly suggest me.
Thanking you in advance.
You tested for mycoplasma contamination? Presence of mycoplasma can hinder transfection efficiency and its presence cannot be detected by traditional indicators/tools such as pH shifts in media and phase contrast microscopy.
Dear Challagandla Anil ,
what kind of nucleic acids are you transfecting?
In our LAP, we transfect mouse embryonic fibroblasts with DNA plasmids and Lipofectamine in the medium we are culuring them (DMEM supplemented with 5% FCS, penicillin (100 U/ml) and streptomycin (100 µg/ml)).
For primary macrophages, transfection with plasmids did not work (either with Lipofectamine nor with other transfection reagents) without inducing massive activation and/or cell death. We managed to transfect them with mRNA and JETmessenger sucessfully (in their cultivation medium, DMEM+10 % FCS).
So the problem you encounter is more likely due to nucleic acid constructs and/or the transfection reagent you use and not due to your medium. Of course there are media that support transfection, but for starters I would stick to the medium, which is used for cell cultivation and instead try different transfection reagents (and use a positive control plasmid that works for transfection in this cell line).
I hope that helps you.
All the best and a good start in the year 2020
Marc
Dear Kiessling,
I haven't checked for mycoplasma's contamination. I will check if the cells are OK, would standard PCR works to detect mycoplasma contamination..?
Thank you.
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