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
- - - - -

stable cell line


  • Please log in to reply
1 reply to this topic

#1 michalcn

michalcn

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 32 posts
0
Neutral

Posted 08 July 2010 - 06:53 PM

Hello,
I transfected phenix cells with pBABE plasmid, collected the virus and infected my cells of interests.
I selected with puramycin until the blank were all dead and collect the cells for RNA and protien.
I expected to see overexpression of my gene of interest but it didn't happen.

I sequence the vector and saw that my gene at least by sequence is there

what are the options for this?
Is there any solution for this problem?
I assumed that my cells are resistance to pura because of the infection but are do you think that they got only the resistance without the constract?

please help!

Thanks
Michal :lol:

#2 laurequillo

laurequillo

    The Goddamn Batman!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 265 posts
1
Neutral

Posted 08 July 2010 - 08:54 PM

Hello,
I transfected phenix cells with pBABE plasmid, collected the virus and infected my cells of interests.
I selected with puramycin until the blank were all dead and collect the cells for RNA and protien.
I expected to see overexpression of my gene of interest but it didn't happen.

I sequence the vector and saw that my gene at least by sequence is there

what are the options for this?
Is there any solution for this problem?
I assumed that my cells are resistance to pura because of the infection but are do you think that they got only the resistance without the constract?

please help!

Thanks
Michal B)


So you couldnt see it nor in RNA neither in protein. Did you check your pBABE in a transient transfection? without any virus, just transfect the plasmid in your cells. I would try that to check if I am able to produce my protein. If not, then I would think there is something wrong with the construct
"He must be very ignorant for he answers every question he is asked" Voltaire

"This is SPARTA!"

"Im the goddamn batman"




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

©1999-2013 Protocol Online, All rights reserved.