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Forward primer not working during DNA sequencing - DNA sequencing (Jan/12/2011 )

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Topo cloning is not directional, so the "forward" direction is aligned with your idea of forward only half the time. You picked a clone with the orientation different from your expectation.

A PCR could work fine with primers that bind to both a template for amplification, and (separately) to a vector region. If you insert the PCR product into that vector, then there will be two copies of the sequence in the resulting plasmid. You will not get interpretable sequencing results using that primer.

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