Abi 3730 l

Cytochrome oxidase I (COI) PCR products were generated by amplification with LCO1490 and HCO2198 primers (Table 2) (Folmer et al. 1994 (link)). Quick-Load Taq 2× Mastermix (New England Biolabs, Ipswich, MA, USA) was used in PCR reactions with total volumes of 25 µl. Amplification protocols were run on MyCycler or S1000 Thermal Cyclers (BioRad, Hercules, California, USA) for these and all other PCR amplifications. PCR reaction conditions were: 95°C for 5 minutes; 35 cycles of 94°C for 1 minute, 46°C for 1 minute, and 72°C for 1.5 minutes; and a final 5 minute extension at 72°C before being placed on a 4°C hold. PCR products were loaded into a 1% agarose gel in TAE buffer, run for 1 hour at 78 V, and visualized using ethidium bromide under ultraviolet light.
PCR products were sequenced using the Sanger dideoxy method as previously described (Borchers & Marcus 2014 ). PCR products were sequenced in both directions with the primers used to generate the products. Sequencing reactions were analyzed on ABI 3130 or ABI 3730×l automated sequencers (Applied Biosystems, Carlsbad, California, USA) and edited using Sequencher 4.6 software (Sequencher 2005 ). Primer sequences were removed leaving sequenced amplified products of 658 bp, which were then aligned in CLUSTAL W version 2.1 (Thompson et al. 1994 (link); Larkin et al. 2007 (link)).

Genomic DNA was extracted using the Promega Wizard Genomic DNA Purification Kit (Promega Biosystems, Sunnyvale, CA, USA). Samples were genotyped at the University of California, San Francisco Genomics Core Facility, using the ABI 3730×l (Applied Biosystems Inc., Foster City, CA, USA). Sequencer DNA Sequence Analysis Software (Gene Codes Corporation, Ann Arbor, MI) was used to analyze the Val66Met alleles.