Although a variety of protocols to isolate high-molecular-weight genomic DNA exist, the isolation and preservation of ultra-high-molecular-weight genomic DNA of sufficient quality and length for error-free third-generation sequencing remains challenging. Inspired by the isolation of high-molecular-weight DNA in agarose plugs suitable to be separated by pulsed-field gel electrophoresis, we report the construction of an incubation chamber for DNA isolation. The chamber is flanked by semi-permeable polycarbonate membranes of variable pore size for the selective diffusion of compounds and components, which allows efficient cell lysis and the subsequent isolation of ultra-high-molecular-weight genomic DNA without shearing. The designed experimental approach is simple, but effective in isolating high-quality ultra-long genomic DNA that is suitable for third-generation sequencing by Oxford Nanopore Technology from challenging bacterial samples. We envisage that genomic DNA isolation using the incubation chamber, or variations thereof, will facilitate the regular sequencing of megabasepair-long DNA fragments, with a multitude of applications in microbiology, immunology, oncology, paleontology and forensic science.