灵活载体克隆
Flexi vector cloning.
作者信息
Blommel Paul G, Martin Peter A, Seder Kory D, Wrobel Russell L, Fox Brian G
机构信息
Center for Eukaryotic Structural Genomics, Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA.
出版信息
Methods Mol Biol. 2009;498:55-73. doi: 10.1007/978-1-59745-196-3_4.
Abstract
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.
摘要
本文描述了一种使用96孔板格式的Flexi载体方法进行连接依赖克隆的方案。完整的方案包括对所需基因进行PCR扩增以附加Flexi载体克隆序列、对PCR产物进行限制性消化、将消化后的PCR产物连接到同样消化的受体载体中、宿主细胞的转化和生长、对转化克隆进行分析以及保存经序列验证的克隆。该方案还包括将经序列验证的克隆转移到另一个Flexi载体质粒骨架中。通过适当调整指定反应体积，可以进行较少数量的克隆反应。
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