Dna extraction

Palacios-Martinez, Iñigo, Jiménez-Ruiz, Yolanda, Otero-Ferre, Pau & García-París, Mario, 2025, Systematics of the genus Dilta Strand, 1911 (Machilidae) in the Canary Islands (NW Africa, Spain) with comments on the phylogeny of Microcoryphia (Insecta), Zoologischer Anzeiger 317 (8), pp. 1-18 : 4

2.2. DNA extraction and sequencing

DNA was extracted from the tissues of 39 specimens, representing a total of 10 genera/subgenera and 23 species of Microcoryphia ( Table 1;

Fig. 1 ). Tissue samples were extracted from the abdominal segments of the specimens preserved in ethanol (96 Ǫ to absolute) and stored at ‒ 20 Ǫ C. Genomic DNA was extracted using the Qiagen DNAtissue extraction kit (Qiagen, Valencia, California, USA).

Polymerase chain reaction (PCR) was used to amplify three DNA fragments: cytochrome oxidase I (cox1) gene fragment with primers LCO1490 ( Folmer et al., 1994) and COI-H ( Machordom et al., 2003); the internal transcribed spacer 2 (ITS2) nuclear region with CAS5p8sFt and CAS28sB1d primers ( Ji et al., 2003); and a fragment of the 18S rDNA (18S) with 18Sai and 18Sbi primers ( Whiting et al., 1997). Polymerase chain reactions were performed in 25 μL, which included 17.8 μL of H 2 O, 2.5 μL of a reaction buffer with MgCl 2 (10 ×), 1 μL of dNTP (10 mM), 1.5 μL of MgCl 2 (50 mM), 0.5 μL of each primer (10 μM), 0.2 μL of Taq polymerase (NZYTech, 5 U/mL) and 1 μL of DNA . The thermal profile of the reactions for cox1 was: initial denaturation at 96 Ǫ C for 5 min, followed by 40 cycles of denaturation at 94 Ǫ C for 30 s, annealing at 42 Ǫ C for 45 s and extension at 72 Ǫ C for 1 min, and a final single extra extension step at 72 Ǫ C for 5 min. For nuclear fragments the thermal profile was: initial denaturation at 96 Ǫ C for 5 min, followed by 40 cycles of denaturation at 94 Ǫ C for 1 min, annealing at 45 Ǫ C (ITS2) or 50 Ǫ C (18S) for 1 min and extension at 72 Ǫ C for 1 min, and a final single extra extension step at 72 Ǫ C for 10 min. After amplification, 3 μL of PCR product was checked by electrophoresis on a 1 % agarose gel. Samples were sent for sequencing to Macrogen Inc (Macrogen Europe, Madrid, Spain). Length of the DNA studied fragments after alignment and trimming comprise a total of 1614 bp: 658 bp of cox1 and 956 bp of 18S, for the 39 newly sampled specimens and 714 bp for the ITS2 for the specimens identified within Dilta ( Table 1).

Figures

Fig. 1. Map of the Iberian Peninsula, Northwestern Africa, Balearic Islands, and Canary Islands, showing the sampling locations (red dots) of sequenced specimens of Microcoryphia, along with the location of additional analyzed GenBank sequences (yellow dots). The base map is a Google Satellite orthophoto. The numbers correspond to the ID of each specimen used in this study (refer to Table 1). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)