Flexi vector cloning.
Flexi vector cloning.
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DOI：
10.1007/978-1-59745-196-3_4
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Fox BG
中科院分区：
文献类型：
--
作者：
Blommel PG;Martin PA;Seder KD;Wrobel RL;Fox BG
描述了一种使用96孔格式的Flexi载体方法进行连接依赖克隆的协议。完整的方法包括：扩增目的基因以添加Flexi载体克隆序列，对PCR产物进行限制性内切酶消化，将消化后的PCR产物连接到类似消化的受体载体中，宿主细胞的转化和生长，转化克隆的分析，以及序列验证克隆的存储。该协议还包括将序列验证的克隆转移到另一个Flexi载体质粒骨架中。通过适当调整所指示的反应体积，可以进行数量较少的克隆反应。
A protocol for ligation-dependent cloning using the Flexi Vector method in a 96-well format is described. The complete protocol includes PCR amplification of the desired gene to append Flexi Vector cloning sequences, restriction digestion of the PCR products, ligation of the digested PCR products into a similarly digested acceptor vector, transformation and growth of host cells, analysis of the transformed clones, and storage of a sequence-verified clone. The protocol also includes transfer of the sequence-verified clones into another Flexi Vector plasmid backbone. Smaller numbers of cloning reactions can be undertaken by appropriate scaling of the indicated reaction volumes.