Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Transfecting primary cells with plasmid


  • Please log in to reply
1 reply to this topic

#1 Aris

Aris

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 67 posts
0
Neutral

Posted 10 June 2010 - 02:26 AM

Hello everyone,

I have been trying for quite some time now to transfect my cells, which are primary vascular rendotelial cells, with a plasmid.
The system I use is Lipofectamin 2000. I transfect the cells and I see the signal in the cells (GFP linked), I propagate the cells,
split them, freeze them down and then thaw them. All this is fine but the problem starts when I thaw them.
For some reason the signal is there in the first couple of hours and then it diminishes rapidly, as if the cells do not express the plasmid any more.
Has anybody seen sth like this before?

#2 Curtis

Curtis

    Metaller Scientist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,112 posts
59
Excellent

Posted 13 June 2010 - 11:31 PM

have you compared this with cells that are not frozen? What is the name of the plasmid you are using?
my boss says that cells degrade GFP inside the host cell specially when the cells are dying.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.