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Transfection & Cilia help!


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#1 SweetT

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Posted 16 August 2011 - 01:45 PM

Hi! I have been trying to overexpress a protein and view whether or not it localizes to the cilia. When I transfect however, none of the transfected cells seem to have a cilia. Cilia seem to disappear when stressed, which may explain why transfection cause them to disapear. I have used turbofect and lipofectamine so far. If anyone has gotten this to work please lend some tips!
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#2 bob1

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Posted 16 August 2011 - 05:42 PM

How long are you leaving them after transfection? Do you change the medium after transfection?

#3 SweetT

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Posted 17 August 2011 - 12:26 PM

What I typically do is use Turbofect (I have also tried lipofectamine and Exgen, but turbofect seems to work best) and add the DNA/turbofect/serum free mix to plates. After 4 hours, I change the media and allow them to grow/express for 48h. I then fix with PFA. Any suggestions?

#4 bob1

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Posted 17 August 2011 - 02:58 PM

I would have expected most of theside effects of transfection to have disappeared after 48 hours. Could you try leaving them for longer than 48 hours?

What is the likelihood that the protein produced off the plasmid is disrupting the cilial formation?




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