John M. S. Bartlett.pdf - Bio-Nica.info

DNA Extraction from Fungi, Yeast, Bacteria 53
13
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 0.04–0.07 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 –20°C 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 4°C.
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
53