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Using a TOPO XL Cloning kit, our lab took some amplified human DNA, inserted it into the vector, and attempted to clone it in E. coli. However, upon sequencing the purified plasmid product, the sequence we got back was E. coli rather the human we had amplified. The primers sat down in the plasmid as intended, just the sequence between the primers appears to be from E. coli (as implied by BLASTing it against a human, getting no results, and then blasting it against E. coli and getting a hit).

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    $\begingroup$ Is it from the E. coli genome, or from the plasmid itself? $\endgroup$
    – iayork
    Mar 8, 2016 at 19:00
  • $\begingroup$ Genome. So it's like (M13F Primer)(E. coli)(M13R Primer) $\endgroup$ Mar 8, 2016 at 21:43
  • $\begingroup$ So you need to troubleshoot. Is the "amplified human DNA" what you think it is? Are you confident in the insertion before the amplification in E. coli? Are you certain you're actually sequencing plasmid? Is there any chance your primers are incorrectly designed, mixed up, or produced wrong by the synthesis company? $\endgroup$
    – iayork
    Mar 9, 2016 at 0:11

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The root cause turned out to be a lying spec. Using a different one, it was revealed that there was next to no template DNA in the initial amplification. No DNA, no good plasmids.

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