Degenerate PCR Primer Amplification of Clam Genes

Primers used were developed by Bowen et al. [37 (link)]. Briefly, degenerate primer pairs developed for the razor clam were used on cDNA from three randomly selected clam samples. Degenerate primer pairs were designed to amplify five genes of interest and two reference genes. The PCR amplifications using these primers were performed on 20 ng of each cDNA sample in 50 μL volumes containing 20–60 pmol of each primer, 40 mM Tris-KOH (pH 8.3), 15 mM KOAc, 3.5 mM Mg (OAc)₂, 3.75 μg/mL bovine serum albumin (BSA), 0.005% Tween-20, 0.005% Nonidet-P40, 200 μM each dNTP, and 5U of Advantage 2^® Taq polymerase (Clontech, Palo Alto, CA, USA). The PCR was performed on an MJ Research PTC-200 thermal cycler (MJ Research, Watertown, MA, USA) and consisted of 1 cycle at 94 °C for 3 min, and then 40 cycles at 94 °C for 30 s, at 60 °C for 30 s, and 72 °C for 2 min, with a final extension step of 72 °C for 10 min. The products of these reactions were electrophoresed on 1.5% agarose gels and resulting bands visualized by ethidium bromide staining. Definitive bands representing PCR products of a predicted base pair size of the targeted gene were excised from the gel and extracted and purified using a commercially available nucleic acid-binding resin (Qiaex II Gel extraction kit, Qiagen, Valencia, CA, USA).