Journal: PLoS ONE
Article Title: A Simple and Effective Method for High Quality Co-Extraction of Genomic DNA and Total RNA from Low Biomass Ectocarpus siliculosus, the Model Brown Alga
Figure Lengend Snippet: Summary of nucleic acids extraction from E. siliculosus brown alga. High yields of good quality DNA and RNA are isolated from as little as 25 Steps 1–5: Harvested tissue is immediately homogenised using commercial 3 mm solid-glass beads in the presence of 1 mL EB containing 100 mM Tris-HCl, 150 mM NaCl, 5 mM DTT and 1% sarkosyl. These stages allow the lysis of the cell wall, the release of highest amount of nucleic acids, the inactivation of cellular nucleases, and the removal of most of the polysaccharides and other insoluble material. Steps 6–10: Simultaneous presence of absolute ethanol and potassium acetate aids polysaccharide precipitation. Moreover proteins, lipids, pigments and cell debris are removed through extraction of the aqueous phase with chloroform. Steps 11–12: Nucleic acids are then recovered by precipitation with 0.8 V of isopropanol and 0.1 V of 3 M sodium acetate (pH 5.2) in the presence of 1% 2-mercaptoetanol at −80°C. During the precipitation step, salts and other solutes are separated from nucleic acids that form a white precipitate collected by centrifugation. The excess of isopropanol and 2-mercaptoetanol are removed through washing the pellet with 75% ethanol. Step 13: All traces of ethanol are removed, the nucleic acid pellet is dried and resuspended in nuclease-free water. After RNase or DNase treatment the superfluous quantities of proteins, polysaccharides, lipids, and cell debris were removed from the extracted DNA and RNA through double extended purification treatment with phenol:chloroform:isoamyl alcohol.
Article Snippet: (a) RNase or DNase treatment To obtain pure DNA, treat aliquots (10–25 µg) of nucleic acid mixtures with 1 µL of RNase A, DNase free enzyme (0.1 mg ml−1 ) (Roche Diagnostic Mannheim, Germany) in a final volume of 100 µL, for 20 min at 37°C.
Techniques: Isolation, Lysis, Centrifugation, Purification