A rapid and cost-effective Chelex-100 resin-based method for genomic DNA extraction from adult zebrafish tissues.

The zebrafish (Danio rerio) is a widely used vertebrate model in biomedical research, yet the rapid and cost-effective extraction of genomic DNA (gDNA) from adult zebrafish tissue remains a practical challenge for routine molecular applications. To address this, we systematically optimized and evaluated a Chelex-100/proteinase K-based extraction protocol across eight adult zebrafish tissues (muscle, liver, heart, spleen, kidney, intestine, brain, and eggs) and compared its performance against that of a commercial kit. The optimized protocol, involving incubation at 56 °C for 15 min and boiling at 100 °C for 10 min, required approximately 25 min per sample. Across all tested tissues (3 biological replicates per tissue), the Chelex-100 method yielded gDNA concentrations ranging from 177.2 ± 3.5 ng/µL to 674.5 ± 22.1 ng/µL per 5 mg of tissue, which were generally higher than those obtained with the commercial kit. However, the purity (A260/A280 ratios) was consistently lower for the Chelex-100 method (1.36-1.83) than for the kit (1.84-2.02), indicating the coextraction of proteinaceous impurities. Despite this, Chelex-extracted gDNA supported successful PCR amplification and Sanger sequencing of target fragments, with consistent amplicon quality. In real-time PCR assays, both methods produced amplification curves, but the Chelex-100 method yielded systematically higher Ct values, suggesting reduced sensitivity with less-pure templates. Crucially, the Chelex-100 method avoided hazardous organic solvents and required no column purification, with an estimated reagent cost of <$0.20 per sample versus approximately $2.00 per sample for the commercial kit. We conclude that this optimized Chelex-100/proteinase K protocol provides a rapid, economical, and operationally simple alternative for routine gDNA extraction from adult zebrafish tissues and is particularly suitable for PCR-based genotyping, sequencing and qPCR applications, though its lower purity warrants careful consideration for high-sensitivity quantitative assays.