Protocol: Nucleic Acid Extraction Method 2024 v8

The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of core procedures: sample preparation and homogenisation, DNA extraction, fragmentation and purification. Detailed protocols are available on protocols.io (Denton et al., 2023b). The fSolSol10 sample was weighed and dissected on dry ice (Jay et al., 2023) and gonad tissue was cryogenically disrupted using the Covaris cryoPREP® Automated Dry Pulverizer (Narváez-Gómez et al., 2023). HMW DNA was extracted using the Automated MagAttract v1 protocol (Sheerin et al., 2023). DNA was sheared into an average fragment size of 12–20 kb in a Megaruptor 3 system (Todorovic et al., 2023). Sheared DNA was purified by solid-phase reversible immobilisation, using AMPure PB beads to eliminate shorter fragments and concentrate the DNA (Strickland et al., 2023). The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system. RNA was extracted from gonad tissue of fSolSol7 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol (do Amaral et al., 2023). The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit. Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
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This work has been supported by the Wellcome Sanger Insitute and Arizona State University.