Hage Aziz El, Houseley Jonathan
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.
Methods Mol Biol. 2013;1054:195-207. doi: 10.1007/978-1-62703-565-1_13.
Pulsed-field gel electrophoresis (PFGE) is a technique that resolves chromosome-sized DNA molecules in an agarose gel. As well as DNA mapping and karyotyping applications, PFGE techniques are well adapted to follow DNA rearrangements over time in a quantitative manner. Because of the very large sizes of the DNA molecules analyzed, DNA preparation, electrophoresis, and Southern blotting processes present unique challenges in PFGE experiments. In this chapter, we describe a robust PFGE protocol covering the preparation of intact Saccharomyces cerevisiae chromosomal DNA, specific running conditions for the resolution of small, medium- and large-sized chromosomes and their by-products, and basic Southern blotting and hybridization instructions for the analysis of these molecules.
脉冲场凝胶电泳（PFGE）是一种在琼脂糖凝胶中分离染色体大小的DNA分子的技术。除了DNA图谱绘制和核型分析应用外，PFGE技术还非常适合以定量方式追踪DNA随时间的重排。由于所分析的DNA分子非常大，DNA制备、电泳和Southern印迹过程在PFGE实验中带来了独特的挑战。在本章中，我们描述了一种可靠的PFGE方案，涵盖完整酿酒酵母染色体DNA的制备、分离小、中、大染色体及其副产物的特定运行条件，以及用于分析这些分子的基本Southern印迹和杂交说明。
