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
- - - - -

problems with PCR confirmation of insert . HELP !


  • Please log in to reply
1 reply to this topic

#1 Debo

Debo

    member

  • Active Members
  • Pip
  • 15 posts
0
Neutral

Posted 22 August 2011 - 12:43 AM

i have to make clones of viral genes l And p . i have made the c dna and amplified with PCR and purified it . then i inserted it in T easy vector and transformed in the host DH5 alpha cells. and i got clones too . in order to confirm the inser i did restriction digestion and i got the exact band desired . further when i did PCR for the particular gene the band is coming below the band for the desired one.its the same for other clones . i am clueless of wat is going on . can anyone help please ?

#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,504 posts
253
Excellent

Posted 22 August 2011 - 03:46 AM

Your primers could be binding to sites other than the ones you intend. There could be repeats in your gene, or incorrect priming due to too low an annealing temperature. I would sequence your construct.




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

©1999-2013 Protocol Online, All rights reserved.