Dneasy blood tissue kit extraction kits

We amplified a 722 base pair (bp) fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene using polymerase chain reaction (PCR), with primers originally designed by Gillis et al. [24 ]. Genomic DNA was extracted from tissue samples of adductor muscle by standard phenol–chloroform methods [57 ] or using QIAGEN DNeasy Blood & Tissue Kit extraction kits following the manufacturer’s protocol. PCR was performed using the following final concentrations in a 20μL reaction: 2mM MgCl₂, 0.8mM dNTPs, 1x PCR buffer, 0.5 μM forward and reverse primers (novel primers [24 ]), 1 unit Taq DNA polymerase, and 50–100 ng DNA template. Cycling conditions were: 95°C for 4 min followed by 35 cycles of 95°C for 30s, 45°C for 30s and 72°C for 30s; final extension period at 72°C for 7 min. PCR products were checked for amplification and size on a 2% agarose gel. All PCR products were purified using exonuclease I and shrimp alkaline phosphatase (ExoSAP; USB, Cleveland, OH, USA) and were submitted for sequencing in both directions to the University of Arizona Genetics Core (Tucson, AZ, USA).