一种制备高分子量酵母DNA的方法。
A method for the preparation of high molecular weight yeast DNA.
作者信息
Lautenberger J A, Chen Z Q
机构信息
Laboratory of Molecular Oncology, National Cancer Institute, Frederick, Maryland 21701-1013.
出版信息
Gene Anal Tech. 1987 Sep-Oct;4(5):87-8. doi: 10.1016/0735-0651(87)90001-x.
A method is described for the isolation of high molecular weight DNA in solution using the principles that have allowed electrophoresis of chromosome-sized DNA in pulse field gradient electrophoresis. Stationary phase yeast cells are converted to spheroplasts by the action of zymolyase in 1 M sorbitol. In the presence of EDTA and sodium lauroyl sarcosinate, proteins are digested with proteinase K. DNA is extracted with phenol and chloroform, and high molecular weight DNA is collected by ethanol precipitation. RNA is removed by RNase digestion of the redissolved pellet, and RNase is removed by chloroform extraction followed by a second ethanol precipitation. The method is rapid and gives a high yield of DNA that is readily digestible by restriction endonucleases.
描述了一种利用在脉冲场梯度电泳中实现染色体大小DNA电泳的原理，在溶液中分离高分子量DNA的方法。通过在1 M山梨醇中zymolyase的作用，将固定相酵母细胞转化为原生质球。在EDTA和月桂酰肌氨酸钠存在下，用蛋白酶K消化蛋白质。用苯酚和氯仿提取DNA，通过乙醇沉淀收集高分子量DNA。通过对重新溶解的沉淀进行RNase消化去除RNA，通过氯仿提取然后进行第二次乙醇沉淀去除RNase。该方法快速，可获得高产率且易于被限制性内切酶消化的DNA。
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