Dna

Haÿ, Vincent, Mennesson, Marion I., Carpentier, Camille, Dahruddin, Hadi, Sauri, Sopian, Limmon, Gino, Wowor, Daisy, Hubert, Nicolas, Keith, Philippe & Lord, Clara, 2025, Phylogeography of Microphis retzii (Bleeker, 1856) and Microphis brachyurus (Bleeker, 1854) in the Pacific, Journal of Fish Biology 106 (2), pp. 602-620 : 604

2.3 | PCR amplification and DNA sequencing

For the specimens fixed in ethanol, DNA was extracted from caudalfin clips using the Macherey and Nagel NucleoSpin 96 Tissues kit following the manufacturer's instructions on an Eppendorf epMotion 5075 robot.

A mitochondrial fragment of the COI gene (650 base pair [bp]) was amplified using the tailed fish specific primers VF2-t1 5 0 - TGTAAAACGACGGCCAGTCAACCAACCACAGACATTGGCAC-3 0; Fis hF2-t1 5 0 -TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCG GCAC-3 0; Fishr2-t1 5 0 -CAGGAAACAGCTATGACACTTCAGGGTGAC CGAAGAATCAGA-3 0 ( Ward et al., 2005); Fr1d-t1 5 0 -CAGGAAACAG CTATGACACCACAGGGTGTCCGARAAYCARAA-3 0 ( Ivanova et al., 2007). DNA was amplified using PCR in a final volume of 20 μL containing 2 μL of buffer, 1 μL of dimethyl sulfoxide (DMSO), 1 μL of bovine albumin serum (BAS), 0.8 μL of deoxynucleotide triphosphates, 0.32 μL of each forward and reverse primer, 0.06 μL of Taq DNA polymerase (Qiagen), 2 μL of DNA , and water. DNA was amplified using a thermal cycler (T100TM Thermal Cycler) after 2 min of denaturation at 94 C followed by 55 cycles (30s, 94 C; 45 s, 54 C; 1 min, 72 C). Successful PCRs were selected on ethidium bromide–stained agarose gels. Sanger sequencing was performed in both directions by Eurofins (http://www.eurofins.fr) using M13 tail primers M13F (21) 5 0 -TGTAAACGACGGCCAGT-3 0; M13R (27) 5 0 -CAGGAAACAGC- TATGAC-3 0 ( Messing, 1983).