Noolandi J, Turmel C
Xerox Research Centre of Canada, Mississauga, Ontario, Canada.
Methods Mol Biol. 1992;12:73-103. doi: 10.1385/0-89603-229-9:73.
The preparation and manipulation of very large DNA molecules for pulsed-field gel electrophoresis (PFGE) requires more care than normally used for smaller molecules. The general protocol used is the preparation of DNA directly in solid agarose blocks (plugs) or beads. Intact cells are encapsidated in agarose (plugs or beads) and are treated with different combinations of enzymes and detergent to remove cell walls, membranes, RNA, proteins, and other materials in order to obtain naked DNA. This is possible because detergent and enzymes can diffuse by Brownian motion through the agarose pores of the plug, whereas the large pieces of DNA cannot and remain sequestered inside the plug. Here, we give detailed protocols of preparation for yeast and mammalian DNA. In addition, we describe the power supplies, switchers, gels, and gel trays required for one-dimensional pulsed-field gel electrophoresis (ODPFGE), as well as the pulse strategies used for the separations.
用于脉冲场凝胶电泳（PFGE）的非常大的DNA分子的制备和操作比通常用于较小分子的操作需要更多的小心。所使用的一般方案是直接在固体琼脂糖块（凝胶块）或珠子中制备DNA。完整的细胞被包裹在琼脂糖（凝胶块或珠子）中，并用不同的酶和去污剂组合处理以去除细胞壁、细胞膜、RNA、蛋白质和其他物质，从而获得裸露的DNA。这是可行的，因为去污剂和酶可以通过布朗运动扩散穿过凝胶块的琼脂糖孔，而大片段DNA则不能并保留在凝胶块内。在这里，我们给出了酵母和哺乳动物DNA制备的详细方案。此外，我们描述了一维脉冲场凝胶电泳（ODPFGE）所需的电源、切换器、凝胶和凝胶托盘，以及用于分离的脉冲策略。
