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
- - - - -

Need help with Blasticidin selection


  • Please log in to reply
1 reply to this topic

#1 grace1115

grace1115

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 03 March 2010 - 10:07 AM

My mammalian cell line of interest is already G418-resistant,so I did a co-transfection with my plasmid of interest (which will confer G418 resistence) along with pCMV-Bsd (will confer blasticidin resistence) at a 10:1 ratio (10ug and 1ug,respectively). Now I am selecting cells with blasticidin resistence (these cells should have taken up both plasmids during the transfection). However, when I was using G418+Blasticidin (2ug/ml) for selection,almost all cells died. So I am wondering if I should do another killing curve (because the G418+Blasticidin killing curve is different from the G418 curve or Blasticidin curve alone?). It seems Blasticidin doesn't work well for my cell line and it's not because my cell grow slowly in the presence of this drug--they just never grow. After 14 days of selection,the cells just died! I don't believe it's because none of them have taken up the pCMV-Bsd plasmid. Has anything similar happened to you guys?

Any input is welcome! thanks!


Grace

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,833 posts
415
Excellent

Posted 03 March 2010 - 03:48 PM

I suspect that the double insult of having G418 and blasticidin selection at one time is the problem. Do one of the plasmids first, select and once grown sufficiently switch it to a maintenance level (half to a 10th of the selective conc) of the selective agent, then transfect the second plasmid.




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

©1999-2013 Protocol Online, All rights reserved.