从阿米茴芹和胡萝卜（胡萝卜属胡萝卜）细胞悬浮培养原生质体中分离脱氧核糖核酸及其性质
Isolation and Properties of Deoxyribonucleic Acid from Protoplasts of Cell Suspension Cultures of Ammi visnaga and Carrot (Daucus carota).
作者信息
Ohyama K, Gamborg O L, Miller R A
机构信息
Prairie Regional Laboratory, National Research Council of Canada, Saskatoon, Saskatchewan S7N OW9, Canada.
出版信息
Plant Physiol. 1972 Sep;50(3):319-21. doi: 10.1104/pp.50.3.319.
A procedure is described for the isolation of native DNA from protoplasts of ammi (Ammi visnaga) and carrot (Daucus carota) cells. Protoplasts were produced from 40 grams of fresh cells by enzyme hydrolysis and lysed with sodium dodecyl sulfate. The DNA was purified by treatment with pronase and ribonuclease. Final isolation was achieved by sucrose density gradient centrifugation.The melting temperature of ammi and carrot DNA in 0.15 m NaCl and 15 mm trisodium citrate buffer, pH 7.0, was 84.0 C and 84.5 C, respectively. The molecular weight for ammi DNA was 1.43 x 10(8), and for carrot DNA it was 1.56 x 10(8). Ammi DNA exhibited a single band at 1.690 grams per cubic centimeter in CsCl, whereas carrot DNA showed two bands, one at 1.693 grams per cubic centimeter and another at 1.706 grams per cubic centimeter. Ammi DNA consisted of a doublestranded form, since denaturation of the DNA caused a complete upward shift of 0.020 grams per cubic centimeter.
本文描述了一种从阿米（Ammi visnaga）和胡萝卜（Daucus carota）细胞原生质体中分离天然DNA的方法。通过酶解从40克新鲜细胞中制备原生质体，并用十二烷基硫酸钠使其裂解。用链霉蛋白酶和核糖核酸酶处理纯化DNA。通过蔗糖密度梯度离心实现最终分离。在pH 7.0的0.15 m NaCl和15 mM柠檬酸钠缓冲液中，阿米和胡萝卜DNA的解链温度分别为84.0℃和84.5℃。阿米DNA的分子量为1.43×10⁸，胡萝卜DNA的分子量为1.56×10⁸。阿米DNA在CsCl中于每立方厘米1.690克处呈现一条带，而胡萝卜DNA显示两条带，一条在每立方厘米1.693克处，另一条在每立方厘米1.706克处。阿米DNA由双链形式组成，因为DNA变性导致每立方厘米完全向上移动0.020克。
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