Publication: Comparison of plasmid dna yield and purity among column-based extraction of different membrane setups
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Date
2026-01
Authors
Helmee, Hazeem
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Abstract
Plasmids, characterized as circular, double-stranded DNA molecules are used for many molecular biology protocols that require high fidelity of DNA integrity. Silica or glass fibre spin columns form the most common method for the purification of plasmids, in terms of speed and reproducibility. However, the expense incurred by commercial kits may limit their use in high throughput applications or in an educational context. Considering that membrane properties affect flow dynamics, clogging tendency and DNA recovery efficiency, systematic control of pore size or column stacking configuration may provide significant variations in plasmid yield and purity. Exploring alternative glass-fibre membrane configurations may offer a more flexible and cost-effective option while still producing DNA suitable for downstream work. This study aimed to compare plasmid DNA yield and purity obtained using self-assembled glass-fibre membrane spin columns with different membrane configurations and to evaluate how consistently each configuration performs when bacterial cell input is reduced. Recombinant E. coli DH10B carrying the pET-14b-EhCL plasmid was revived and mass-cultured to generate cell pellets. Plasmid DNA was purified using columns assembled with different glass-fibre membrane setups (80/22 μm, 80/45 μm, and 80/45/22 μm), with an 80/80 μm configuration used as the control reference. DNA concentration and purity were assessed by spectrophotometry using A260/A280 and A260/A230 ratios. Differences in yield and purity among configurations were analysed using one-way ANOVA (P < 0.05). To examine performance across varying input, serial dilutions (2X to 0.125X) were tested and evaluated using linear regression (R²) to describe each setup’s dynamic profile range. Plasmid yield differed across membrane configurations. The 80/80 μm setup produced the highest yield and was significantly higher than 80/45 μm and 80/45/22 μm. The 80/45/22 μm configuration also outperformed 80/45 μm, while 80/22 μm yielded higher than 80/45 μm. For A260/A280, all configurations were within or close to the expected DNA purity range and did not differ significantly. In contrast, A260/A230 showed significant differences involving the 80/22 μm setup compared with 80/45 μm and 80/45/22 μm. Across serial dilutions, the most stable linear yield trend was observed for 80/45/22 μm (R² = 0.924), followed by 80/80 (R² = 0.8556), 80/22 (R² = 0.7823), and 80/45 μm (R² = 0.6871). Overall, glass-fibre configuration influenced plasmid DNA yield and affected A260/A230 purity more noticeably than A260/A280. The 80/80 μm setup delivered the strongest yield, while the 80/45/22 μm arrangement showed the most consistent yield scaling across different input levels. These findings suggest membrane stacking can be tailored depending on whether maximum yield or more predictable performance across variable samples is the priority.