Hello
I've been transfecting HeLa cells with an rtTA plasmid that contains GFP and my protein of interest. I use Xfect kit from clontech (it's polymer based. you transfect, change medium after 4h to overnight, and then I add the dox and after few hours to 72 hours I do my experiments, the incubation time with dox depends on the experiment).
It worked fine until 2 weeks ago when it stopped working. No GFP stain. I've tried to troubleshoot it- I thawed a new vial of cells, made new medium (it's DMEM with glutamine, Na pyruvate, 10%FBS and penn strep), changed to a tet proven FBS, made new fresh doxycycline. I'm not sure if it's a new vial of polymer from the transfection kit, but I'm using the same kit (same catalog number) as before, and also called clontech to double check there were no changes. I tried to increase the dose of dox, and check GFP after 24, 48 and 72 hours after dox administration, but I don't get the GFP stain I used to get only 2 weeks ago. The plasmid is rtTA, promoter is CMV. and it's the same plasmid DNA I used when it worked. I checked the pDNA on nanodrop and it looks ok, ratios around 2.
Anyone had the same problem? Any suggestions on what the problem might be?
Thanks!
Two suggestions:
1. Try to synchronize the cells.
2. Transfect the cells with some other plasmid containing gfp or rfp.
A silly question but is your UV mercury lamp working alright? Maybe it needs replacement.
Hi Syed
It's not a lamp issue because we first detected the problem when running experiments with the transfected cells and visualizing them with a confocal microscope and it was pretty clear there that our GFP+ cells diminished significantly.
How do you recommend synchronizing the cells? with serum free medium? for an hour and then change the medium to serum rich for transfection?
Thanks
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