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!

Colonies present but not positive

Kan resistant insert TOPO

  • Please log in to reply
1 reply to this topic

#1 pmggms2



  • Active Members
  • Pip
  • 11 posts

Posted 01 August 2014 - 01:17 PM

Ok, so I have been working with three plant genotypes of the same species and I am also working with three different inserts; gene 1 is 1.7Kb, gene 2 is 3.1Kb and gene 3 is 4.8Kb. I have placed all three genes into TOP 10 vector for sequencing in all three genotypes. While doing so I was able to get gene 1 in all three genotypes sequenced, but for gene #2 I picked colonies from my plate and placed them onto a master plate and did colony PCR for all three genotypes. Now here is my issue I have screened my colonies and have positive colonies for genotypes 1 and 3 but for genotype 2. I end up with a few colonies but none of them are positive. I have repeated this cloning process multiple times for this particular insert and am getting frustrated. The same issue is for gene three where I have positive colonies for genotype 1 and 2 but not #3.  When I did phusion PCR with cDNA I received amplifications for gene 2 and cannot seem to understand why they are not positive. PLEASE HELP!!!!!!!!! 

#2 bob1


    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 6,706 posts

Posted 01 August 2014 - 01:53 PM

Top 10 is a strain of E.coli, not a vector, which backbone are they on?


Are you using the same PCR primers for all 3 genotypes?


What are the components of your primers?


Which restriction enzymes are you using?


How competent are your cells?


Have you tried to verify insertions by methods other than PCR?

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

©1999-2013 Protocol Online, All rights reserved.