The standard procedure in the lab for plasmid isolation usually involves a 2-mL, 16 h over-night cultivation in 15-mL bioreaction tubes in LB medium. This is time consuming, and not suitable for high-throughput applications. This study shows that it is possible to produce plasmid DNA (pDNA) in a 1.5-mL microcentrifuge tube with only 100 μL cultivation volume in less than 7 h with a simple protocol. Compared with the standard LB cultivation for pDNA production reaching a final pDNA concentration range of 1.5–4 μg mL(–1), a 6- to 10-fold increase in plasmid concentration (from 10 up to 25 μg mL(–1) cultivation volume) is achieved using an optimized medium with an internal substrate delivery system (EnBase®). Different strains, plasmids, and the applicability of different inoculation tools (i.e. different starting ODs) were compared, demonstrating the robustness of the system. Additionally, dissolved oxygen was monitored in real time online, indicating that under optimized conditions oxygen limitation can be avoided. We developed a simple protocol with a significantly decreased procedure time, enabling simultaneous handling of more samples, while a consistent quality and a higher final pDNA concentration are ensured.