Nucleosomes are substrates for a broad range of factors, including those involved in transcription or chromosome maintenance/reorganization and enzymes that covalently modify histones. Given the heterogeneous nature of nucleosomes in vivo (i.e., varying histone composition, post-translational modifications, DNA sequence register), understanding the specificity and activities of chromatin-interacting factors has required in vitro studies using well-defined nucleosome substrates. Here, we provide detailed methods for large-scale PCR preparation of DNA, assembly of nucleosomes from purified DNA and histones, and purification of DNA and mononucleosomes. Such production of well-defined nucleosomes for biochemical and biophysical studies is key for studying numerous proteins and protein complexes that bind and/or alter nucleosomes and for revealing inherent characteristics of nucleosomes. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Large-scale PCR amplification of DNA Basic Protocol 2: DNA and nucleosome purification using a Bio-Rad Mini Prep Cell/Prep Cell Basic Protocol 3: Nucleosome reconstitution via linear gradient salt dialysis.