Orlando S J, Nabavi M, Gharakhanian E
Department of Biological Sciences, California State University Long Beach, 1250 Bellflower Blvd, Long Beach, CA 90840, USA.
J Virol Methods. 2000 Nov;90(2):109-14. doi: 10.1016/s0166-0934(00)00176-2.
A rapid method for the small-scale isolation of SV40 virions and SV40 DNA is presented. CV-1 monkey epithelial cells are transfected with linear SV40 DNA. After the onset of transfection, cells are lysed by several freeze/thaw cycles and virions are isolated using polyethylene glycol (PEG) precipitation of DNase I treated lysates. Viral DNA is released by proteinase K and dithiothreitol treatment of the isolated virions followed by phenol/chloroform extraction and ethanol precipitation. This method yields on average 7.5x10(4) plaque forming units (PFUs) and DNA of adequate purity and concentration to be used for restriction analysis on ethidium bromide agarose gels from a single 35-mm tissue culture dish.
本文介绍了一种小规模分离SV40病毒粒子和SV40 DNA的快速方法。用线性SV40 DNA转染CV-1猴上皮细胞。转染开始后，通过多次冻融循环裂解细胞，并用聚乙二醇（PEG）沉淀经DNase I处理的裂解物来分离病毒粒子。通过蛋白酶K和二硫苏糖醇处理分离出的病毒粒子，然后进行酚/氯仿萃取和乙醇沉淀来释放病毒DNA。该方法平均可从单个35毫米组织培养皿中获得7.5×10⁴个噬斑形成单位（PFU）以及纯度和浓度足以用于在溴化乙锭琼脂糖凝胶上进行限制性分析的DNA。
