Lockett T J
CSIRO Division of Biotechnology, Laboratory for Molecular Biology, North Ryde, New South Wales, Australia.
Anal Biochem. 1990 Mar;185(2):230-4. doi: 10.1016/0003-2697(90)90284-g.
A method for the efficient preparation of high quality bacteriophage lambda DNA from cleared lysates is described. Advantages of the method include high DNA yields (typically around 0.8 micrograms of DNA/1 ml of cleared lysate), speed of processing (approximately 2 h from lysate to DNA), economy, and the absence of any requirement for phenol or chloroform extractions. The technique involves the concentration of phage particles by standard polyethylene glycol precipitation followed by enzymatic treatment to remove contaminating RNA and DNA. Phage particles are then lysed with sodium dodecyl sulfate (SDS) at elevated pH and temperature. Contaminating protein/SDS complexes are rendered insoluble by the addition of potassium acetate and removed by centrifugation. The quality of the resultant DNA is comparable to that prepared by cesium chloride banding for all standard molecular biological purposes providing that spermidine is included in all restriction endonucleases digestions.
本文描述了一种从澄清裂解物中高效制备高质量噬菌体λDNA的方法。该方法的优点包括DNA产量高（通常约为0.8微克DNA/1毫升澄清裂解物）、处理速度快（从裂解物到DNA约2小时）、经济实惠，且无需进行任何酚或氯仿提取。该技术包括通过标准聚乙二醇沉淀浓缩噬菌体颗粒，然后进行酶处理以去除污染的RNA和DNA。接着在升高的pH值和温度下用十二烷基硫酸钠（SDS）裂解噬菌体颗粒。通过添加醋酸钾使污染的蛋白质/SDS复合物不溶，并通过离心去除。只要在所有限制性内切酶消化中都加入亚精胺，所得DNA的质量就与通过氯化铯梯度离心制备的DNA质量相当，可用于所有标准分子生物学目的。
