DNA Extraction from Fungi, Yeast, and Bacteria (David Stirling)

1. Introduction Although individual microorganisms may well require a unique DNA extraction procedure, here we include robust techniques for the preparation of DNA from fungi, yeast, and bacteria, which yield DNA suitable for a PCR template. 2. Materials 2.1. Fungal Extraction
1. CTAB extraction buffer: 0.1 M Tris-HCl, pH 7.5, 1% CTAB (mixed hexadecyl trimethyl ammonium bromide), 0.7 M NaCl, 10 mM EDTA, 1% 2-mercaptoethanol. Add proteinase K to a final concentration of 0.3 mg/mL prior to use. 2. Chloroformisoamyl alcohol (241).

2.2. Yeast Extraction

1. Yeast extraction buffer A: 2% Triton X-100, 1% sodium dodecyl sulphate, 100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0. Phenolchloroformisoamyl alcohol: Phenol is presaturated with 10 mM Tris-HCl, pH 7.5. Prepare a mixture of 25241 phenolchloroformisoamyl alcohol (v/v/v). This solution can be stored at room temperature for up to 6 mo, shielded from light. 2. Glass beads. Diameter range 0.040.07 mm (Jencons Scientific Ltd, UK), suspended as 500 mg/mL slurry in distiller water. 3. Ammonium acetate (4 M).

2.3. Bacterial DNA Protocol

1. Lysozyme/RNase mixture: 10 mg/mL lysozyme, 1 mg/mL RNase, 50 mM Tris-HCl (pH 8.0). Store at 20C in small aliquots. Do not refreeze after thawing. 2. STET: 8% sucrose, 5% Triton X-100, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA, pH 8.0. 3. Filter sterilize and store at 4C.

3. Methods 3.1. Fungal Protocol

1. Grind 0.2 to 0.5 g (dry weight) of lyophilized mycellar pad in a mortar and pestle. Transfer to a 50-mL disposable centrifuge tube. 2. Add 10 mL (for a 0.5 g pad) of CTAB extraction buffer. 3. Gently mix to wet all the powdered pad. 4. Place in 65C water bath for 30 min. 5. Cool and add an equal volume of chloroform/isoamyl alcohol (241). 6. Mix and centrifuge at 2000g for 10 min at room temperature. 7. Transfer aqueous supernatant to a new tube. 8. Add an equal volume of isopropanol. 9. High molecular weight DNA should precipitate upon mixing and can be spooled out with a glass rod or hook. 10. Rinse the spooled DNA with 70% ethanol. 11. Air dry, add 1 to 5 mL of TE containing 20 g/ mL RNAse A. To resuspend the samples, place in 65C bath or allow pellets to resuspend overnight at 4C.

3.2. Yeast Protocol

1. Collect cells from fresh 5 mL culture by centrifugation at 2000g for 10 min and resuspend in 0.5 mL of water. 2. Transfer cells to 1.5-mL microfuge tube and collect by centrifugation at 15,000g for 10 min, pour off supernatant and resuspend in residual liquid. 3. Add 0.2 mL of buffer A, 200 L of glass beads, and 0.2 mL of phenolchloroformisoamyl alcohol (25241). 4. Vortex for 3 min and add 0.2 mL of TE. 5. Centrifuge at 15,000g for 5 min and then transfer aqueous to new tube. 6. Add 1 mL of 100% EtOH (room temperature), invert tube to mix, and centrifuge at 15,000g for 2 min. 7. Discard supernatant and resuspend pellet in 0.4 mL of TE (no need to dry pellet). 8. Add 10 L of 4 M ammonium acetate, mix, and then add 1 mL of 100% EtOH and mix. 9. Centrifuge at 15,000g for 2 min and dry pellet. Resuspend in 50 L of TE.

3.3. Bacterial DNA Protocol

1. Collect the bacteria from a 15-mL overnight culture into a 1.5-mL microfuge tube. 2. Resuspend pellet with 300 L of STET buffer and add 30 L of RNAse/lysozyme mixture. 3. Boil for 1 min 15 s. 4. Centrifuge at 15,000g for at least 15 min. 5. Take supernatant and phenol extract with 150 L of STET-saturated phenol. 6. Spin and take supernatant. Add 1/10 volume 4 M lithium chloride (autoclaved). Let sit on ice for 5 to 10 min. 7. Spin and take supernatant. Add equal volume isopropanol at room temperature and incubate for 5 min. 8. Centrifuge at 15,000g for at least 15 min. No pellet will be visible. 9. IMPORTANT: wash with 80% ethanol (95% will cause the residual Triton to precipitate). 10. Resuspend pellet in 50 to 200 L of TE.