Cold Spring Harb Protoc. 2021 Jan 4;2021(1):2021/1/pdb.prot101766. doi: 10.1101/pdb.prot101766.
Thin (0.4-1.5 mm) polyacrylamide-urea gels provide high resolution of RNAs up to 1000 nt in size and are capable of resolving single-stranded fragments of RNA that differ in length by as little as 1 nt. The polyacrylamide gel is cast between two glass plates that are separated by two thin Teflon or nylon spacers. A so-called shark's tooth comb or, less frequently, a standard slotted comb forms the sample wells into which the RNA samples are loaded before electrophoresis. In contrast to electrophoresis using agarose gels, which occurs while the gel is horizontal, polyacrylamide gels are run while in the vertical position. Gels are also typically run at 45°C-55°C, which is the melting temperature of RNA, and in the presence of 6-8 m urea. The gel recipe and protocol presented here for 8 m urea/TBE polyacrylamide gels can be used for a variety of applications including mapping RNA with nuclease S1, ribonuclease protection assay, or analysis of RNA by primer extension.
薄（0.4-1.5 毫米）聚丙酰胺-尿素凝胶可将大小达 1000 个核苷酸的 RNA 进行高分辨率分离，并且能够分辨相差仅 1 个核苷酸的单链 RNA 片段。聚丙酰胺凝胶浇铸在两块玻璃平板之间，两块玻璃平板由两个薄聚四氟乙烯或尼龙间隔物隔开。一种所谓的鲨鱼齿梳（有时也用标准的有槽梳）将样品孔形成于其中，在电泳之前将 RNA 样品加载到这些孔中。与在琼脂糖凝胶中进行的水平电泳不同，聚丙酰胺凝胶是在垂直位置下运行的。凝胶通常在 45°C-55°C 下运行，这是 RNA 的融解温度，并在存在 6-8 m 尿素的情况下运行。这里介绍的用于 8 m 尿素/TBE 聚丙酰胺凝胶的凝胶配方和方案可用于多种应用，包括用核酸酶 S1、核糖核酸酶保护分析或引物延伸分析对 RNA 进行作图。
