Polymerase Chain Reaction
Mitochondrial DNA- Cytochrome b
The entire (~1140 bp) cytochrome b gene was amplified in PCR using universal primers (L14724, Irwin & Kocher, 1991; H15910, Oshida, Lin, Masuda, & Yoshida, 2000) for the tissue sample (HSU 8180). In the museum specimens, an approximately 300 bp region of the mitochondrial cytochrome b gene was amplified with the primer L14724 (Irwin & Kocher, 1991) and a newly designed reverse primer, GOR_R1, from a G. o. californicus GenBank sequence (Accession #: AF063060) using Primer3 (Untergasser et al., 2012). Fragments were mapped to published sequences and visualized in Geneious Prime v 2020.0.4 (Kearse et al., 2012). Primers were ordered from IDT at 35.4 nmol concentration, solubilized, and diluted to 100 µM prior to PCR. Singleplexed PCR was performed in 16 µl reactions containing 2.0 µl of DNA template, 4.5 µl of ddH2O, 0.5 µl of each primer, and 8.5 µl of DreamTaq Green PCR Master Mix (Thermo Fisher Scientific Inc., Waltham, MA) with the following thermocycler profile: 95°C for 1 min; 30 cycles (tissues) or 35 cycles (museum samples) of 95°C for 30 sec, 45°C for 30 sec, 72°C for 30 sec; 72°C for 5 mins. All amplifications were performed on either an Applied Biosystems MiniAmp or BioRad T-100 cycler. Successful PCR amplifications were replicated twice in each sample to ensure validity, and a 1-1.5% agarose gel was run to visualize PCR products.