Properties of an in vitro selected Pb2+ cleavage motif.
Properties of an in vitro selected Pb2+ cleavage motif.
复制标题
体外选择的 Pb2 裂解基序的特性。
DOI：
10.1021/bi00198a023
复制
发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Uhlenbeck,OC
中科院分区：
文献类型：
--
作者：
Pan,T;Dichtl,B;Uhlenbeck,OC
材料与方法rna的制备及活性测定。使用T7 RNA聚合酶（Milligan & Uhlenbeck, 1989）对合成的寡脱氧核苷酸模板进行体外转录合成所有RNA，并用变性聚丙烯酰胺凝胶纯化。为了检测RNA的裂解，1µRNA在15 mM MOPS中，1 pH 7.0，在85℃下加热2分钟，冷却到22℃，加入适当浓度的MgCh（通常为10 mM）。为了启动裂解反应，将新制备的Pb (OAc) 2加入到所需的浓度，并将反应混合物在25℃下孵育，等量的反应混合物被去除，并通过加入等体积的9 M尿素/50 mM来停止反应
MATERIALS AND METHODSPreparation of RNAs and Activity Assays. All RNAs were synthesized by in vitrotranscription of synthetic oligodeoxynucleotide templates using T7 RNA polymerase (Milligan & Uhlenbeck, 1989) and purified on denaturing polyacrylamide gels. To assay RNAs for cleavage, 1 µ RNA in 15 mM MOPS, 1 pH 7.0, was heated at 85 C for 2 min and cooled to 22 C and MgCh was added to the appropriate concentra-tion (usually 10 mM). To initiate the cleavage reaction, freshly prepared Pb (OAc) 2 was added to the desired concentration and the reaction mixture was incubated at 25 C. Aliquots of reaction mixtures were removed, and the reaction was stopped byaddition of an equal volume of 9 M urea/50 mM