A centrifuge-free, plate-based protocol to prepare chemically competent Escherichia coli.

Classical calcium-chloride protocols for Escherichia coli transformation achieve high efficiency but rely on equipment (refrigerated centrifuges, shakers, turbidity control) that is often unavailable in teaching or resource-limited laboratories. We developed a centrifuge-free, plate-based workflow for preparing chemically competent E. coli and systematically optimized its key parameters. On solid LB medium, DH5α reached exponential growth by 2 h post re-streak and remained log-phase until 6 h; harvesting at 2 h yielded the highest competence. Buffer titrations identified 0.25 M CaCl₂ with 15% (v/v) glycerol as optimal. For post-shock outgrowth, SOC consistently outperformed LB and 0.5 × LB. Transformation displayed a DNA-dose dependence: 1-0.1 ng supercoiled plasmid per 100 μL reaction maximized efficiency. The simplified CaCl₂/glycerol buffer produced 105-106 transformants per μg DNA, sufficient for routine cloning. Temperature-dependency experiments revealed that on-ice DNA-cell co-incubation is the only essential low-temperature step; competent cells can be prepared at room temperature and the heat-shock step may be omitted without loss of efficiency. This streamlined protocol reduces equipment and time requirements while delivering reproducible performance, making it well suited for teaching laboratories and other low-resource settings.