Denaturation and electrophoresis of RNA with formaldehyde.
Denaturation and electrophoresis of RNA with formaldehyde.
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DOI：
10.1101/pdb.prot080994
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发表时间：
2015-02-02
影响因子：
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通讯作者：
Rio, Donald C
中科院分区：
文献类型：
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作者：
Rio, Donald C
电泳大小分级可用于在含甲醛的琼脂糖凝胶上变性和分离大mRNA分子（0.5-10 kb）。甲醛含有羰基，可与鸟嘌呤、腺嘌呤和胞嘧啶的亚氨基或氨基反应形成席夫碱。这些共价加合物阻止正常的碱基配对，并使RNA保持变性状态。因为这些加合物不稳定，所以甲醛必须存在于凝胶中以保持RNA处于变性状态。该方案描述了用甲醛制备琼脂糖凝胶及其在水平电泳装置中的设置。制备RNA样品，并在甲酰胺和甲醛溶液中变性，用0.5- 10-kb大小的标记物通过凝胶进行电泳。电泳后，使用几种不同类型的染色剂之一对凝胶进行染色以使RNA标记物或rRNA可视化。
Electrophoretic size fractionation can be used to denature and separate large mRNA molecules (0.5-10 kb) on formaldehyde-containing agarose gels. Formaldehyde contains a carbonyl group that reacts to form Schiff bases with the imino or amino groups of guanine, adenine, and cytosine. These covalent adducts prevent normal base pairing and maintain the RNA in a denatured state. Because these adducts are unstable, formaldehyde must be present in the gel to maintain the RNA in the denatured state. This protocol describes the preparation of an agarose gel with formaldehyde and its setup in a horizontal electrophoresis apparatus. RNA samples are prepared and denatured in a solution of formamide and formaldehyde and, with 0.5- to 10-kb size markers, subjected to electrophoresis through the gel. Following electrophoresis, the gel is stained to visualize RNA markers or rRNA using one of several different types of stains.