Miniprep Isolation and Restriction Mapping of Plasmid pBR322 Isolated from Escherichia coli: Experimental and Bioinformatic Validation

Abstract Plasmid DNA isolation and restriction mapping remain foundational techniques in molecular biology, biotechnology, and genetic engineering. Among classical cloning vectors, pBR322 continues to serve as a benchmark plasmid for teaching, method standardization, and comparative molecular analysis due to its well-characterized genetic architecture and antibiotic resistance markers. The present study focuses on the mini-preparation (mini-prep) isolation of plasmid pBR322 from Escherichia coli, followed by experimental restriction digestion and bioinformatic validation to generate and confirm its restriction map. Plasmid DNA was isolated using an alkaline lysis- based mini-prep protocol optimized to minimize shearing and ensure high molecular integrity. The purified plasmid was subjected to restriction digestion using the endonucleases HincII and PvuII, individually and in combination, and analyzed through agarose gel electrophoresis. The electrophoretic profiles revealed plasmid bands corresponding to the expected size range of approximately 4.3 kb, confirming successful isolation. Restriction digestion results demonstrated that HincII acts as a double-cutter enzyme on pBR322, while PvuII exhibits single-site or nicking activity, consistent with known sequence data. Experimental observations were further validated using in silico restriction analysis tools, including NCBI databases and NEB Cutter, which corroborated the predicted cleavage sites and fragment sizes. The study highlights the reliability of combining experimental restriction analysis with bioinformatic tools for accurate plasmid characterization. These findings reinforce the continued relevance of pBR322 as a model plasmid and demonstrate a robust, reproducible approach for plasmid mapping applicable in academic teaching laboratories and preliminary molecular cloning workflows. Keywords: Plasmid DNA, pBR322, Mini-prep, Restriction mapping, Agarose gel electrophoresis, Bioinformatics validation