SIMPLIFIED MAMMALIAN DNA ISOLATION PROCEDURE
SIMPLIFIED MAMMALIAN DNA ISOLATION PROCEDURE
复制标题
DOI：
10.1093/nar/19.15.4293
复制
发表时间：
1991-08-11
影响因子：
14.9
通讯作者：
BERNS, A
中科院分区：
文献类型：
--
作者：
LAIRD, PW;ZIJDERVELD, A;BERNS, A
结果和讨论我们通常发现DNA产率超过使用苯酚/氯仿提取和乙醇沉淀获得的分离物的产率。一般来说，样品含有很少的RNA。DNA样品的质量足以使用各种限制性内切酶进行Southern印迹分析，包括仅在低盐条件下有活性的酶。我们已经用这种方法分析了高达20 kb的片段。我们在限制酶消化中加入0.1 mg BSA/ml，以吸收任何残留的SDS、蛋白酶或其他抑制成分。上述程序目前在几个小鼠遗传学实验室中常规使用。所涉及的减少的手工劳动被认为是该方法的重要益处。当偶尔遇到问题时，这是由于组织样品太大、裂解期间搅拌不足或DNA溶解期间搅拌不足。如果遇到限制性消化的问题，酚/氯仿提取后再沉淀的选择仍然是开放的。
RESULTS AND DISCUSSION We generally find the DNA yields to exceed those of isolates obtained using phenol/chloroform extractions and ethanol precipitation. In general the samples contain very little RNA. The DNA samples are of sufficient quality for Southern blot analysis using a variety of restriction enzymes, including those active only in low salt conditions. We have used this method to analyze fragments of up to 20 kb. We include 0.1 mg BSA/ml in the restriction enzyme digestion to absorb any residual SDS, proteases, or other inhibitory elements. The procedure described above is now being used routinely in several mouse genetics laboratories. The reduced manual labor involved is perceived as an important benefit of the method. When problems were occasionally encountered, this was due to either too large a tissue sample, insufficient agitation during lysis, or insufficient agitation during dissolution of the DNA. If problems with restriction digestion are encountered, the option of a phenol/chloroform extraction followed by a reprecipitation is still open.