Sbyr1safe

Genomic DNA was extracted from the sampled tissue from each individual following the protocol described in Martín-Torrijos et al. [39 (link)]. Mitochondrial 16S and COI genes were used as markers in this study for detecting intraspecific genetic variation in crayfish. The primers pair used to amplify the mitochondrial 16S gene, 1472 [54 (link)] and Tor12sc [55 (link)], amplified a fragment that included partial sequences of the 12S rRNA, the 16S and the val-tRNA. The primer pairs used to amplify the mitochondrial COI gene was C/N 2769 [56 (link)] and LCO1490 [57 ]. Both primers pairs were used in a single round PCR following the protocols in Matallanas et al. [38 (link)], including negative and positive controls. Three microliters of aliquots of the amplification products were analyzed to check positive amplicons in 1% agarose TAE gels stained with SBYR1Safe (Thermo Fisher Scientific, Waltham, MA, USA). The amplified products were purified using a QIAquick PCR Purification Kit (Qiagen, Germany). Double-stranded PCR products were sequenced using an automated sequencer (Macrogen, Spain). Sequences were assembled and edited using the program Geneious v6.14 8 [58 (link)], and a BLAST search was run on each sequence to confirm they belonged to WCC species.

Genomic DNA was extracted from the sampled tissue from each individual following the protocol described in Martín-Torrijos et al. [39 (link)]. Mitochondrial 16S and COI genes were used as markers in this study for detecting intraspecific genetic variation in crayfish. The primers pair used to amplify the mitochondrial 16S gene, 1472 [54 (link)] and Tor12sc [55 (link)], amplified a fragment that included partial sequences of the 12S rRNA, the 16S and the val-tRNA. The primer pairs used to amplify the mitochondrial COI gene was C/N 2769 [56 (link)] and LCO1490 [57 ]. Both primers pairs were used in a single round PCR following the protocols in Matallanas et al. [38 (link)], including negative and positive controls. Three microliters of aliquots of the amplification products were analyzed to check positive amplicons in 1% agarose TAE gels stained with SBYR1Safe (Thermo Fisher Scientific, Waltham, MA, USA). The amplified products were purified using a QIAquick PCR Purification Kit (Qiagen, Germany). Double-stranded PCR products were sequenced using an automated sequencer (Macrogen, Spain). Sequences were assembled and edited using the program Geneious v6.14 8 [58 (link)], and a BLAST search was run on each sequence to confirm they belonged to WCC species.