Propagating Gateway Vectors.
Propagating Gateway Vectors.
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传播网关向量。
DOI：
10.1101/pdb.prot094920
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发表时间：
2018
影响因子：
--
通讯作者：
Walhout,AlberthaJM
中科院分区：
文献类型：
--
作者：
Reece-Hoyes,JohnS;Walhout,AlberthaJM
产生用于Gateway重组克隆系统的入口和目的地载体的储备需要将它们转化到大肠杆菌菌株DB3中。1，因为该菌株对Gateway盒中携带的ccdB基因的影响具有免疫力，所以它们可以在其中复制。然而，ccdB基因中的突变可能以低频率出现，并且这些突变质粒将因此允许E. coli（如DH5α）。因此，在制备Gateway质粒的新储备物后，它们在E.必须检测大肠杆菌。这涉及获得多个载体储备，每个载体储备产生于在单个DB3中生长的单个质粒。1个细菌菌落，并将每种原液转化到两种DB3中。1和E.大肠杆菌中进行。Gateway克隆反应中仅应使用能有效杀死标准克隆菌株的载体储备液（即转化后未获得菌落或仅获得少量菌落）。该序列可以在3D中执行。
Generating stocks of Entry and Destination vectors for use in the Gateway recombinatorial cloning system requires transforming them into Escherichia coli strain DB3. 1, where they can replicate because this strain is immune to the effects of the ccdB gene carried in the Gateway cassette. However, mutations in the ccdB gene can arise at low frequency, and these mutant plasmids will consequently allow growth of standard cloning strains of E. coli (eg, DH5α). Therefore, after making new stocks of Gateway plasmids, their ability to grow in cloning strains of E. coli must be tested. This involves obtaining multiple stocks of vector, each arising from a single plasmid grown in a single DB3. 1 bacterial colony, and transforming each stock into both DB3. 1 and the preferred cloning strain of E. coli in a controlled fashion. Only vector stocks that effectively kill the standard cloning strain (ie, no or few colonies are obtained after transformation) should be used in Gateway cloning reactions. The sequence can be performed in 3 d.