Applied biosystems 3130 dna analyser

Sequencing reactions were performed in both directions using a Big Dye Terminator cycle sequencing kit v3.1 (Applied Biosystems, Foster City, CA, USA), and the same set of PCR primers. Each sequencing reaction was carried in a total volume of 10 μL containing 1 μL of purified PCR product, 0.5 μL of Big Dye^® Terminator v3.1 Ready Reaction Mix (PE Applied Biosystems), 1× sequencing buffer (PE Applied Biosystems), and 3.6 pmol of F or R primer, and the remaining volume consisted of ultrapure water. The sequencing products were purified with Sephadex G50 columns (GE Healthcare) and analyzed in an Applied Biosystems 3130 DNA Analyser (PE Applied Biosystems, Warrington, UK). Sequences were edited using the Sequencher software v. 5.2.4 (Genes Codes Corporation, Ann Arbor, MI, USA) and the primer regions were removed.

Sequencing reactions proceeded in both directions using a Big Dye Terminator cycle sequencing kit v3.1 (Applied Biosystems, Foster City, CA, USA) and Applied Biosystems 3130 DNA Analyzer (Applied Biosystems). Sequencing reactions were carried out with the same set of PCR primers. The sequencing products were purified using Sephadex G50 columns (GE Healthcare) and analysed in an Applied Biosystems 3130 DNA Analyser (PE Applied Biosystems). Sequences were edited in Sequencher v.4.9 software (Genes Codes Corporation, Ann Arbor, MI, USA), and the primer regions removed. In addition to these novel sequences, 162 sequences from GenBank were also included, to serve as references in the phylogenetic and species delimitation analysis. The COI gene sequences were aligned first by nucleotides using the Muscle algorithm [48 (link)] implemented in SeaView [49 (link)], and then by amino acid using TranslatorX [50 (link)].