Anamika Kumari, Vinay Bhushan Kumar and Jyoti Pandey

A reliable and cost-effective CTAB-based DNA extraction protocol has been developed for milky mushroom. Advanced molecular genetic analysis relies heavily on techniques such as phylogenetic analysis, marker-assisted selection, purity analysis, gene mapping, DNA fingerprinting, and heterotic analysis. However, labour-intensive and time-consuming nature of high-throughput DNA isolation remains a critical bottleneck scenario. This study presents a modified CTAB-DNA isolation, Sodium acetate and Ammonium acetate precipitation and centrifugation effectively removed proteins and other impurities. This method that is simplified, cost-effective, and adaptable to basic laboratory settings. Notably, this approach eliminates the need for liquid nitrogen and hazardous chemicals such as 2BM, enhancing laboratory safety and reducing costs. The protocol effectively overcomes challenges posed by non-cellulosic components in mushroom cell walls. The isolated DNA demonstrated high quality DNA, with absorbance at A260/A280 ratios ranging from 1.7 to 1.9, and was free of contaminants. This modified method offers a rapid, easy, and reliable solution for mushroom research, facilitating molecular genetic studies. Successful amplification using random primer OPN-01 further validated the efficacy of the protocol. This standardized method offers a practical solution for molecular biology applications, including RAPD, SSR, restriction digestion, Southern blot, and cloning techniques such as phylogenetic analysis (Kumari et al., 2023), marker-assisted selection (Kumar et al., 2023), diversity analysis (Singh et al., 2022; Singh et al., 2023), gene mapping, and DNA fingerprinting (Suman et al., 2018), heterotic analysis (Raaj et al., 2018) play a vital role, without using liquid nitrogen or toxic chemicals. Key words: DNA isolation, milky mushroom, Calocybe indica, CTAB, Liquid nitrogen and βmercaptoethanol.

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