Optimized plasmid extraction through controlled temperature, prolonged alkaline lysis, and gentle mixing.

Alkaline lysis serves as an effective technique for plasmid extraction. However, its widespread adoption is significantly impeded by inconsistencies in key technical parameters across different documents. To address these issues, we conducted a rheological behavior analysis to understand the pattern of cell lysis and performed a series of plasmid extraction experiments to determine the optimal technical parameters. The result showed that gentle mixing (inverting tube five times, I5)), extended lysis time (10 min), and elevated lysis temperature (25 °C), each produced the best plasmid preparations, based on plasmid yield, quality, and performance in downstream molecular biology assays. We also discovered that bacterial density influenced cell lysis process. Extending the alkaline lysis duration neither induced resistance to restriction endonuclease (RE) digestion of plasmids nor significantly compromised the integrity of plasmids and host genome; instead, it enhanced the plasmid release. High-frequency and small-amplitude mixing tended to generate more genomic fragments, whereas low-frequency and large-amplitude mixing was likely to result in a higher occurrence of open circular (OC) plasmids. Bacterial lysis at low temperatures (4 °C) could lead to genomic contamination and reduce lysis efficiency. Overall, these findings underscored the importance of technical details and provided guidance for future plasmid extractions.