A new device‑mediated miniprep method

Abstract

Small-scale plasmid DNA preparation or miniprep is a fundamental technique in estimation cloning experiments and is widely used for DNA methylation analysis in epigenetic research. Current plasmid DNA minipreps use the alkali-SDS-based method in a three-solution format and require spin column-based purification steps. This procedure requires the vortexing or pipetting of pelleted bacteria by centrifugation and manual mixing of the solutions. Here, we describe a centrifuge/mixer-based instrument with the ability to perform centrifugation, vibration, and rotor oscillation in order to perform all steps of plasmid DNA isolation by device only. We found that by applying rotor oscillation-driven mixing of solutions added in the lysis and neutralization steps, homogeneous mixing was achieved within 5 s at a rotor oscillation amplitude of 45 degrees and oscillation frequency of 400 +/- 30 rpm, yielding the maximal quantity and quality of plasmid DNA. No increase in host chromosome presence purified by this approach occurs for high-copy-number plasmids compared to manually performed miniprep, and indeed, there is a significant decrease in the presence of the chromosomal fraction in low-copy-number plasmids. The supercoiled form of plasmid DNA purified at a rotor oscillation amplitude of 45 degrees does not turn into an open circular (OC) isoform when the plasmid is stored for 1 year at plus four degrees, in contrast to the plasmid purified with rotor oscillation amplitudes of 270 degrees, 180 degrees and 90 degrees. The programmed time-work-efficient protocol of plasmid miniprep installed in the device gives the extreme simplicity of plasmid minipreps speeding up and facilitating the isolation of plasmid DNAs. Keypoints New devise-mediated plasmid miniprep method (DM) performs all mixing steps without operator intervention. The DM method produces plasmid DNAs free of the dCCC form and significantly reduces the contamination with genomic DNA in the low-copy-number plasmid. DM miniprep plasmids are reliable templates for bisulfite PCR sequencing analysis.