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In Topic: RNAi problem - Gene is knocked down, but not protein
24 August 2012 - 11:57 AM
Ambinlab, on 17 January 2012 - 02:37 PM, said:
Dear All,
After spending more than 3 weeks, I got my cloning working. I got three plasmids with the right sequences which (should) express shRNA against my gene of interest.
I transfected 293s with my plasmids and was able to see the GFP, which was a marker on the vector. SO I am pretty sure that the cells were transfected efficiently.
I harvested them after 48h and 72h, but I could not see any knockdown effect on western blot.
After spending more than 3 weeks, I got my cloning working. I got three plasmids with the right sequences which (should) express shRNA against my gene of interest.
I transfected 293s with my plasmids and was able to see the GFP, which was a marker on the vector. SO I am pretty sure that the cells were transfected efficiently.
I harvested them after 48h and 72h, but I could not see any knockdown effect on western blot.
If you have your vector with your GOI and GFp expressing sequence both of them together, what might have happened is, when your plasmid broke during insertion, it might have destroyed your GOI sequence. I had similar kind of problem with those vector, after transfection and keeping the cells in selection for more than two weeks, there were still plenty of cells in my 6 well plate, but the cells that were showing green in GFP were very few.. I continued my experiment and picked up the colonies (I was unsure which colonies to pick) the non green colonies showed expresiion of my GOI but cells from green colonies were just like my regular cells. i used RT PCR for initial screening...
Nabin
