Dna

Atherton, Sarah, 2025, A new species of Sisubiotus (Tardigrada: Macrobiotidae) from northern Sweden with a key to the genus, Zootaxa 5679 (3), pp. 399-418 : 401

DNA extraction, amplification, and sequencing

DNA from whole individual adults or eggs was extracted using a Qiagen DNeasy kit following the manufacturer’s instructions. PCR amplification was performed using 0.2 ml PuReTaq Ready-to-go PCR Beads (GE Healthcare) with 5 pmol each forward and reverse primers and 3 µl DNA .

Three gene regions were selected for amplification: the ~650 bp “Folmer region” of the mitochondrial cytochrome oxidase subunit 1 (COI), a ~850 bp fragment including the D2 region of the ribosomal large subunit (28S), and a ~1400 bp fragment of the ribosomal small subunit (18S). Table 1 lists all primer pairs and PCR protocols. PCR-product was examined on 1% agarose gel with gel-green and purified using ExoSAP-IT enzymes (Exonuclease and Shrimp Alkaline Phosphotase; GE Healthcare) and DNA sequencing was conducted by Macrogen Europe ( Netherlands).