Smith D R
Molecular Neurobiology Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, Republic of Singapore.
Methods Mol Biol. 1993;18:433-8. doi: 10.1385/0-89603-245-0:433.
After digestion of DNA with a restriction enzyme (Chapter 50), it is usually necessary, for both preparative and analytical purposes, to separate and visualize the products. In most cases, where the products are between 200 and 20,000 bp long, this is achieved by agarose gel electrophoresis. Agarose is a linear polymer that is extracted from seaweed and sold as a white powder. The powder is melted in buffer and allowed to cool, whereby the agarose forms a gel by hydrogen bonding. The hardened matrix contains pores, the size of which depends on the concentration of agarose. The concentration of agarose is referred to as a percentage of agarose to volume of buffer (w/v), and agarose gels are normally in the range of 0.3 to 3%. Many different apparatus arrangements have been devised to run agarose gels; for example, they can be run horizontally or vertically, and the current can be conducted by wicks or the buffer solution. However, today, the "submarine" gel system is almost universally used. In this method, the agarose gel is formed on a supporting plate, and then the plate is submerged into a tank containing a suitable electrophoresis buffer. Wells are preformed in the agarose gel with the aid of a "comb" that is inserted into the cooling agarose before the agarose has gelled. Into these wells are loaded the sample to be analyzed, which has been mixed with a dense solution (a loading buffer) to ensure that the sample sinks into the wells.
用限制性内切酶消化DNA后（第50章），出于制备和分析目的，通常都需要分离并观察产物。在大多数情况下，产物长度在200至20,000 bp之间，这可通过琼脂糖凝胶电泳来实现。琼脂糖是一种线性聚合物，从海藻中提取，呈白色粉末状出售。将粉末在缓冲液中熔化，然后冷却，琼脂糖通过氢键形成凝胶。硬化的基质含有孔隙，孔隙大小取决于琼脂糖的浓度。琼脂糖浓度以琼脂糖占缓冲液体积的百分比（w/v）表示，琼脂糖凝胶的浓度通常在0.3%至3%之间。为进行琼脂糖凝胶电泳设计了许多不同的装置；例如，可以水平或垂直运行，电流可通过灯芯或缓冲溶液传导。然而，如今几乎普遍使用“潜水”凝胶系统。在这种方法中，琼脂糖凝胶在支撑板上形成，然后将板浸入装有合适电泳缓冲液的槽中。在琼脂糖凝胶中借助“梳子”预先形成孔，在琼脂糖凝胶化之前将梳子插入冷却的琼脂糖中。将与浓溶液（上样缓冲液）混合的待分析样品加入这些孔中，以确保样品沉入孔中。
