PCR results of 16S rDNA and D-Loop region amplification. Yellow circles indicate samples subsequently submitted for sequencing. A 1 kb ladder was used. Note the smear signature in the majority of larval PCRs.

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Context 1

... for 45 s; (3) final extension: 72°C for 5 min. PCR products were visualized on a 1.8% agarose gel with a 1 kb ladder to assess yield. Amplification of mtDNA from the larvae showed evidence of DNA damage that can be seen in the smear signatures of the majority of larval samples compared to the absence of such signatures in the positive controls (Fig. 2a,b). Thus, only a portion of the larvae from which DNA was extracted produced enough amplified mtDNA fragments to proceed with sequencing. The D-Loop region amplified somewhat more successfully than the 16S rDNA, possibly due to its shorter length (349 bp versus 568 bp). The damage prevented a series of detailed population analyses that ...

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... 568 bp). The damage prevented a series of detailed population analyses that were initially planned; nevertheless, the species identification by Brooks and Goetz (2014) could still be tested. Six WRS larvae from six different tows and five ERS larvae were used that showed clear bands in the post-PCR gel in both the 16S rDNA and D-Loop region PCRs (Fig. 2a,b). Two of the positive controls (one adult from the Weddell Sea and one larva from the Ross Sea, Table 1) were also included for sequencing. To prepare for Sanger sequencing, unincorporated dNTPs and primers were removed from PCR products using a pre-sequencing kit (Affymetrix) according to the manufacturers instructions. Fragments were ...