Isolation of DNA from yeasts.
Isolation of DNA from yeasts.
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从酵母中分离 DNA。
DOI：
10.1016/0003-2697(89)90360-6
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发表时间：
1989
影响因子：
2.9
通讯作者：
Jonathan Jeffery
中科院分区：
文献类型：
--
作者：
William Mann;Jonathan Jeffery
描述了允许从广泛不同的酵母（产朊假丝酵母、酿酒酵母和粟酒裂殖酵母）制备DNA的方法。该方法具有可靠的重现性，并且获得的DNA具有用于构建基因文库的适当质量（大小范围的上限始终为50-150 kbp）。在方法A中，通过用来自哈茨木霉的高度纯化的酶混合物处理将酵母细胞转化成原生质球，将原生质球在月桂酰肌氨酸盐/EDTA缓冲液中裂解，并将裂解物与蛋白酶K孵育，然后通过三氟乙酸铯梯度直接离心。通过乙醇沉淀从适当的级分中回收DNA，并将再溶解的沉淀物与核糖核酸酶一起孵育。对于其余的分离，给出了两个方案，一个避免，一个包括苯酚/氯仿萃取。以这种方式，可以获得大小高达约150 kbp的DNA。在方法B中，不制备原生质球。通过在液氮下研磨破碎酵母细胞，然后以类似于方法A，方案2的方式进行后处理。后续步骤取决于需要DNA的目的。蔗糖或盐密度梯度离心或琼脂糖凝胶电泳的传统方法适用于大小选择。碘化钠/硅胶基质技术可以快速有效地从琼脂糖凝胶中回收DNA。
Methods are described that allow DNA to be prepared from widely different yeasts (Candida utilis, Saccharomyces cerevisiae, and Schizosaccharomyces pombe). The methods are reliably reproducible, and the DNA obtained is of appropriate quality for the construction of gene libraries (upper limit of size range consistently 50–150 kbp). In method A, yeast cells are converted into spheroplasts by treatment with a highly purified mixture of enzymes from Trichoderma harzianum, the spheroplasts are lysed in a lauroylsarcosinate/EDTA buffer, and the lysate is incubated with proteinase K and then directly centrifuged through a cesium trifluoroacetate gradient. DNA is recovered from the appropriate fractions by ethanol precipitation, and the redissolved precipitate is incubated with ribonuclease. For the rest of the isolation, two protocols are given, one avoiding and one including phenol/chloroform extraction. In this way, DNA up to about 150 kbp in size can be obtained. In method B, spheroplasts are not made. Yeast cells are broken by grinding under liquid nitrogen and are then worked up in a manner similar to method A, protocol 2. Subsequent steps depend on the purpose for which the DNA is required. Traditional methods of sucrose or salt density gradient centrifugation or agarose gel electrophoresis are applicable for size selection. A sodium iodide/silica matrix technique allows fast and effective DNA recovery from agarose gels.
在三氟乙酸铯梯度中同时显带大鼠胚胎 DNA、RNA 和蛋白质。
影响因子：
2.9
作者：
Mirkes,PE
通讯作者：
Mirkes,PE
影响因子：
3.5
作者：
C. Holm;Douglas Meeks-Wagner;W. L. Fangman;D. Botstein
通讯作者：
C. Holm;Douglas Meeks-Wagner;W. L. Fangman;D. Botstein