ABSTRACT
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.
Subject(s)
DNA/genetics , High-Throughput Screening Assays/methods , Homologous Recombination/genetics , Saccharomyces cerevisiae/genetics , Cloning, Molecular/methods , Genetic Vectors/genetics , Plasmids/genetics , Polymerase Chain Reaction/methods , Synthetic Biology/methodsABSTRACT
High-throughput, inexpensive DNA sequencing is an essential component of large-scale DNA assembly operations. Using traditional and acoustic liquid-handling robotics, Illumina's Nextera Tagmentation reactions can be miniaturized and paired with custom PCR index primers to produce highly multiplexed NGS libraries for pooled sequencing. This chapter describes a high-throughput protocol that enables the simultaneous sequencing of thousands of DNA constructs in a single sequencing run at a dramatically reduced cost compared to bench-top methods.
