Reference Work Entry

Encyclopedia of Genetics, Genomics, Proteomics and Informatics

pp 523-523

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DNA Extraction

There are a large number of procedures applicable to the major taxonomic groups. Only some basic outlines can be provided here. BACTERIA: 1. Grow bacteria in culture medium of choice to a high density (one to several days). 2. Harvest cells by centrifugation. 3. Lyse cell pellet in TE buffer (Tris-EDTA) containing SDS (sodium dodecyl sulfate) and proteinase K (free of DNase) at 37 °C for an hour. 4. Make it 0.5 M for NaCl and add CTAB (cetyl trimethylammonium chloride) to precipitate cell wall, polysaccharides, proteins, etc. but keep DNA in solution. 5. Add equal volume of chloroform:isoamyl alcohol and centrifuge to remove CTAB and polysaccharides. 6. Save supernatant. 7. Remove protein by phenol:chloroform:isoamyl alcohol and centrifuge. 8. From supernatant precipitate DNA by isopropanol. 9. Wash the precipitated DNA with 70% ethanol. 10. Take up DNA in TE buffer and store it in refrigerator. PLANTS: 1. Use 10–50 g clean young tissue (from plants which have been kept in dark for 2 days to reduce starch). 2. Grind it to powder in liquid nitrogen. 3. Extract DNA in pH 8 Tris-EDTA buffer containing a detergent (SDS or Sarkosyl) for about 1 to 2 h at 55°C. 4. Pellet debris by centrifugation at 4 °C (6,000 rpm, 10 min) and collect supernatant DNA. 5. Precipitate DNA from supernatant by 0.6 volume cold isopropanol (−20°C) by centrifugation (8,000–10,000 rpm, 4°C, 15 min). 6. Pellet is taken up in TE buffer. MAMMALIAN TISSUES: 1. Tissue or cell pellet (0.2–1 g) washed clean, and powdered in frozen liquid nitrogen. 2. Lysis of cells (in NaCl, Tris buffer, EDTA, SDS, pH 8, proteinase K). 3. Extraction of DNA in phenol:chloroform:isoamyl alcohol and centrifuge (10 min, 10,000 rpm). 4. To supernatant add 0.5 vol. 7.5 M ammonium acetate and 2 vol. cold ethanol to precipitate DNA by centrifugation (2 min, 5,000 rpm). 5. Wash DNA by 70% ethanol. 6. Suspend DNA in TE buffer. All of the above procedures are very similar. Further purification may be necessary by ultracentrifugation in CsCl. Quantity may be determined on the basis of absorption of ultraviolet light of 260 nm (quartz cuvettes) in a spectrophotometer. In a reasonably pure DNA preparation the ratio of OD260: OD280 is about 2:1. One OD is about 50 μg/mL for double-stranded DNA and 40 μg/mL for single-stranded DNA; 1 pg DNA is about 6.5 × 1011 Da. centrifuge, ultracentrifuge, spectrophotomete​r, SDS, Sarkosyl, TE buffer, EDTA, CTAB, Tris, proteinase K

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