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Colonies present but not positive

Kan resistant insert TOPO

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#1 pmggms2

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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

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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?







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