Reference Work Entry

Encyclopedia of Genetics, Genomics, Proteomics and Informatics

pp 523-523

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 d ...

This is an excerpt from the content