Hi everyone,
I tried many times to transfect my fibroblast with my plasmid, but so far, it never worked.
I tried to vary different factors such as
1) Day of treatment (24 to 72 hours)
2) Different cell types (WS-1 and NHDF)
3) Different concentration of Lipofectamine3000
4) Different concentration of plasmid (1, 5, 10, 20, 50 ng plasmid)
5) Different concentration of P300 (specific reagent for Lipofectamine3000)
After treatment, I control for transfection efficiency (thanks to eGFP). I always have a little amount of "green cells" (expressing the plasmid) - but only 4-5 cells/microscopy field.
Real-time PCR indicates an increase of 2 fold of my protein of interest.
Here are the details you may need:
Cells
1) WS-1 (fibroblasts, cell line)
2) NHDF (fibroblasts, primary cell)
Plasmid
pTracer plasmid, with CMV promoter, my gene of interest, EM7promoter/eGFP/BlasR with a AmpR promoter and gene
Transfection reagent
Lipofectamine3000
Protocol
Lipofectamin3000, manufacturer´s protocol with personal optimization (see above)
Note
When I prepare my plasmid dilution, I sometime observe a white precipitate, like a net, impossible to resuspend.
The question is: where is the problem? What could I change?
I am really thankful for all the advise you may provide !
Regards,
Greg
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