Evolution of the RNA coliphages: the role of secondary structures during RNA replication.
Evolution of the RNA coliphages: the role of secondary structures during RNA replication.
复制标题
RNA 大肠杆菌噬菌体的进化：RNA 复制过程中二级结构的作用。
DOI：
10.1101/sqb.1987.052.01.037
复制
发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Mills,DR
中科院分区：
文献类型：
--
作者：
Priano,C;Kramer,FR;Mills,DR
METHODSGeneralprocedures。根据Eoyang和August（1971）的方法，从感染Qfl的大肠杆菌Q13噬菌体中分离出Qfl复制酶，省略了羟基磷灰石步骤。RNA的熔化是通过在指定的溶液中加热到100~ 3分钟，然后在冰浴中旋转迅速冷却到0~来进行的。苯酚提取的方法是将等体积的苯酚水溶液（用0.1%的8-羟基喹啉[Mallinckrodt]和50 mM Tris-HC1在pH为7.8时饱和）混合，然后离心分离相。从含有400 mM NaC1或2 M乙酸铵的溶液中沉淀RNA，加入2.5体积的无水乙醇，在16℃（如果存在NaCI）下冷却60分钟，或在0℃（如果存在乙酸铵）下冷却15分钟，然后离心浓缩RNA。RNA微球用70%乙醇洗涤，真空干燥。
METHODSGeneralprocedures. Qfl replicase was isolated from bacteriophage Qfl-infected E. coli Q13 according to the method of Eoyang and August (1971), with the hydroxyapatite step omitted. Melting of RNA was carried out by heating to 100~ for 3 minutes in a specified solution and then chilling rapidly to 0~ by swirling in an ice bath. Phenol extraction was carried out by mixing equal volumes of an aqueous solution with phenol (saturated with 0.1% 8-hydroxyquinoline [Mallinckrodt] and 50 mM Tris-HC1 at pH 7.8) and then separating phases by centrifugation. Precipitation of RNA from solutions containing either 400 mM NaC1 or 2 M ammonium acetate was carried out by adding 2.5 volumes of absolute ethanol, chilling the solution for either 60 minutes at-16~(if NaCI was present) or 15 minutes at 0~(if ammonium acetate was present), and then concentrating the RNA by centrifugation. RNA pellets were washed with 70% ethanol and dried in a vacuum.