12S PCR Metabarcoding Protocol for Fish Detection in Estuarine Samples v1

This methodology has been developed for the use of MiFish-U and MiFish-E primers multiplexed with Platinum SuperFi II (2X) and Zymo clean up under a touchdown PCR program, aimed at the specific amplification of fish species from estuarine samples with inhibitors. The primers, originally developed by Miya et al., 2015, and Kawato et al., 2021, target the hypervariable region of the mitochondrial DNA 12S rRNA gene in fish species including Elasmobranchii. Platinum SuperFi II (2X) is a high fidelity polymerase and is well suited for multiplexing as it can work using primers with different melting temperatures at 60°C.