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
- - - - -

Mutation screening


  • Please log in to reply
1 reply to this topic

#1 Elena Velo

Elena Velo

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 07 August 2014 - 11:50 PM

Dear all,

 

I was wondering if someone would willing to help me. At our lab, we obtained mice with point mutation of the gene of our interest, which is activated and stabilized by double strand breaks (DSB), but I do not see anything on Western blot. My project is to find out if in our mice is the mutation have some phenotype, if our gene is activated.  I cultivated MEFs and irradiated by 5, 8, 10, 15, 20 Gy, but Western blot is without result. I cannot see difference between WT and Mutant irradiated MEFs.

 

Does someone have experience how to find out if the mutation occurs? Does someone immortalized MEFs? Do you know any technique how to distinguish the mutation (point mutation) in genome?

 

I just want to know if the point mutation in our mice have/will have some phenotype.

 

Thank you for your time. I really appreciate it.

Elena



#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,669 posts
396
Excellent

Posted 08 August 2014 - 03:16 AM

More detail needed...

 

Is this a western that has worked previously (on mouse cells too?)? Did you have a positive control for the protein on your western? 

 

Is the mutation Mendelian inherited? How do you know that the MEFs you cultivated were positive (+/+ or +/- not -/-) for the mutation? Can yo PCR the locus?  If so dHPLC might work for genotyping. Possibly HRM analysis too.






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

©1999-2013 Protocol Online, All rights reserved.