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LARGE-SCALE LEAF DNA EXTRACTION
- Funnel lyophilized, ground leaf tissue into a 25 ml capacity Oakridge tube.
- Soak in 10 mL CTAB extraction buffer (2% CTAB, 100 mM Tris-HCl pH 8, 20 mM EDTA pH 8, 1.4 M NaCl, Proteinase K to 0.125 mg/ml) at 65°C for 3 hours. (Add the Proteinase K immediately prior to incubation.) Gently vortex samples to mix.
- Spin for 10' at 4000 rpm. Remove 5-8 ml to a fresh tube.
- Extract with 5-8 ml 24:1 chloroform/isoamyl alcohol. Vortex thoroughly.
- Centrifuge 15' at 4000 rpm.
- Remove top layer (approximately 8-12 ml) to another tube. Add 2/3 volume isopropanol, mix, and incubate 10 minutes at RT.
- Centrifuge 4000 rpm for 15'.
- Pour off isopropanol and add 5 ml 70% ethanol.
- Spin at 4000 rpm for 15'. Pour off ethanol and dry for 30' at 80°C.
- Depending on pellet sizw, resuspend DNA in anywhere from 1-5 ml TE (10 mM Tris-HCl pH 8, 1 mM EDTA pH 8) or dH20. Allow to sit overnight at 4°C to completely dissolve pellet.
Reference
Kang, et al. Plant Molecular Biology Reporter 16: 1-9.
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