2026-10-14T14:17:31Z https://api.figshare.com/v2/oai

oai:figshare.com:article/33939643 2026-09-18T17:50:52Z category_4 category_7 category_13 category_14 category_21 category_272 category_873 category_734 category_931 portal_5 item_type_3 month_year_09_2026

<p>Comparison of the two gDNA extraction methods.</p> Wei Feng (225281) Boya Li (3147633) Yiqiong Niu (25073545) Qiannan Xu (230981) Xiandun Zhai (23757735) Biochemistry Medicine Genetics Molecular Biology Biotechnology Environmental Sciences not elsewhere classified Chemical Sciences not elsewhere classified Biological Sciences not elsewhere classified Information Systems not elsewhere classified suggesting reduced sensitivity operationally simple alternative danio rerio </ consistent amplicon quality 56 ° c 02 ), indicating 00 per sample routine molecular applications 5 ± 22 84 – 2 2 ± 3 time pcr assays div >< p adult zebrafish tissues 100 ° c 36 – 1 estimated reagent cost routine gdna extraction genomic dna extraction genomic dna effective extraction zebrafish (< tested tissues qpcr applications 5 ng target fragments systematically optimized pure templates proteinase k proteinaceous impurities practical challenge particularly suitable optimized protocol involving incubation generally higher consistently lower column purification biomedical research based method based genotyping a280 ratios <$ 0 15 min 10 min <div><p>The zebrafish (<i>Danio rerio</i>) 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.</p></div> 2026-09-18T17:50:41Z Dataset Dataset 10.1371/journal.pone.0358591.t002 https://figshare.com/articles/dataset/_p_Comparison_of_the_two_gDNA_extraction_methods_p_/33939643 CC BY 4.0