Amyris Inc., Emeryville, CA, USA.
Methods Mol Biol. 2020;2205:79-89. doi: 10.1007/978-1-0716-0908-8_5.
Yeast homologous recombination is a reliable, low-cost, and efficient method for DNA assembly. Using homology regions as short as 24 base pairs, constructs of up to 12 unique parts can be assembled into a diverse range of vectors. The simplicity and robustness of this protocol make it amenable to laboratory automation and high-throughput operations. Here we describe a high-throughput protocol to generate DNA parts through PCR, assemble them into a vector via yeast transformation, and "shuttle" the resulting plasmid constructs into E. coli for storage and propagation. Though this protocol is intended for high-throughput workflows, it can be easily adapted for bench-scale DNA assembly.
酵母同源重组是一种可靠、低成本、高效率的 DNA 组装方法。使用短至 24 个碱基的同源区域，可以将多达 12 个独特的部分组装成多种不同的载体。该方案的简单性和稳健性使其适用于实验室自动化和高通量操作。在这里，我们描述了一种通过 PCR 生成 DNA 片段、通过酵母转化将它们组装到载体中、然后将得到的质粒构建体“穿梭”到大肠杆菌中进行存储和繁殖的高通量方案。尽管该方案旨在用于高通量工作流程，但它也可以很容易地适应于台式 DNA 组装。
