Dna barcoding

Tump, Marloes, Vonk, Ronald, Burridge, Alice K., Beentjes, Kevin K., Goetze, Erica & Peijnenburg, Katja T. C. A., 2025, Genetic diversity of hyperiid amphipods along a meridional transect in the Atlantic Ocean, Contributions to Zoology 94 (3), pp. 261-291 : 265

DNA barcoding

DNA was extracted from tissues using NucleoMag B-Beads and binding buffer MB2 using the magnetic separator NucleoMag SEP. A ~685 base pair fragment of the mitochondrial cytochrome oxidase subunit 1(CO1) gene was amplified from each specimen. Polymerase Chain Reaction (PCR) amplifications included PCR buffer CL 2.5 μL (Qiagen), MgCl₂ (25 mM) 0.5 μL, BSA (100mM) 0.5 μL, dNTP (2.5 mM) 0.5 μL, Qiagen Tag (5 U/ μL) 0.25 μL, primers M13F–jgLCO1490F (10 pMol/ μL) 1 μL and M13F-jgHCO2198R (10 pMol/ μL) 1 μL, and 2.0 μL of template DNA . PCR was performed with primers jgLCO1490F and jgHCO2198R ( Geller et al., 2013), which were tailed with M13F and M13R for sequencing ( Messing 1983). PCR conditions were 94 °C for 3 min of denaturation, followed by 40 cycles of 94 °C for 15 sec, 50 °C for 30 sec, 72 °C for 40 sec, and a final extension of 72 °C for 5 minutes. Sequencing of forward and reverse strands was carried out by Baseclear B.V. (Leiden, the Netherlands).