Extraction of nucleic acids from lyophilized plant material.
Four methods for extracting nucleic acids from lyophilized cotton (Gossypium hirsutum L. cv. Stoneville 62) leaves and roots were compared. They were based on the use of: (I) HC10(4); (II) KOH; (III) a mixture of 90% phenol, Tris (hydroxymethyl) aminomethane buffer, and sodium lauryl sulfate; and (IV) NaCl. (I) extracted large amounts of RNA but little DNA and extracted much carbohydrate and protein contaminants. (II) gave a good yield of both RNA and DNA but extracted such large amounts of contaminating material that purification of RNA on an anion exchange column was necessary. (III) extracted only part of the RNA and practically no DNA, but extracted contaminating materials. (IV) resulted in high yields of both RNA and DNA when modified to omit preliminary acid extraction of impurities. The use of cold trichloroacetic acid instead of ethanol, to precipitate NaCl-extracted nucleic acids, separated the nucleic acids from most of the carbohydrate and acid-soluble phosphate contaminants and resulted in good agreement among results by ultraviolet absorbance, pentose tests, and phosphate analysis. This method also resulted in lower protein contents and better ultraviolet absorption spectra than the other methods tested. Nucleic acids were extracted from leaves of 14 other species of plants, in addition to cotton, by this modified NaCl procedure.
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Crops Research Division, Agricultural Research Service, United States Department of Agriculture and the Oklahoma Agricultural Experiment Station, Stillwater, Oklahoma.