plese provide the protocol how we can go through transfection of the cell using the GFP tag etc, positive control of the expression system to check the expression of the channel?
I will tell you what we do for 293T cells. You need 293T cells at 60-80% confluence on the day of transfection. You have to mix 10ug of GFP-coding plasmid with 40-60ul of PEI (Polyethylenimine 1mg/ml) depending of the size of your plasmid in 1 ml serum free medium feverything must be at room temperature), for a 10cm dish of 293 cells. 293T cells medium should have been changed the same day (without passing). Let the mix of plasmid+PEI stand for 15-30 min at room temp. And then add it carefully on the 293t Cells the petri dish drop after drop. You change the medium 5-16hrs post-transfection and observe fluorescence 24-72hrs after. I hope this helps.
Hi Jitendra,
Alternatively to Natacha transfection procedure (which by the way works perfectly fine), you can simply use the old fashion calcium/phosphate transfection procedure - it cost nothing nd works just fine with those cells. You can just google the protocol, and ask me and I will send it to you.
Dear Dr.Singh,
HEK 293 cells are relatively good in terms of transfection compare to other cells. Use of Rotifect as transfection reagent might be an easy option to transfect GFP tagged channels. I have attached its procedure however, calcium-phosphate method also work very well and relatively cost effective. Best wishes.
thanks to all for help
Weiss thanks can you send me protocol even priveltly to me.
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