If sanger results include nucleotides past my reverse primer does that imply I am amplifying unwanted regions?

12 December 2014 1 3K Report

I sequenced with both the forward and reverse primers in separate reactions (one tube forward primer + amplified DNA, and the other tube reverse primer + amplified DNA). The reverse reaction shows only the forward primer and part of my expected amplicon. The forward reaction shows the expected amplicon, the reverse primer, and an extra nucleotide sequence that does not match the template sequence. I BLASTed the extra ending sequence and it did not have any hits. Can I confirm I am amplifying my desired PCR product?

Molly Resnick

David,

Thank you for your response. I amplified from genomic DNA and my amplicons are rather short (>200 base pairs).

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