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Why do I see multiple bands after PCR amplification using the Decode negative selection primers?

We see multiple bands which could represent hairpins with longer barcodes than the predicted 250bp amplicon since on average the barcodes are 60nt but range up to 120nt long. The upper band is about 350bp and is the major PCR product in most cases following amplification of gDNA. What we think is happening is that the amplicons from this pooled population have a high degree of sequence homology and only differ in the relatively short regions of the barcode. Therefore, duplexes can form between two strands of unique barcodes. We believe that these hybrid duplexes have secondary structure which alters the speed at which the amplicon migrates through a gel. We do not see the 350bp band with gDNA from cells transduced with GIPZ control hairpins because in this case we are not amplifying a mixed population of hairpins, but a single hairpin. We have sequenced both amplicons (250bp and 350bp) that are obtained from the gDNA and confirmed that they are both valid and therefore, both bands should be isolated from the gel.

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