Magnetic, Microplate-Format Plasmid Isolation Protocol for High-Yield, Sequencing-Grade DNA. (October 2000)

• Record Type: Journal Article
• Authors: Skowronski, E.W.; Armstrong, N.; Andersen, G.; Macht, M.; McCready, P.M.
• Is Part Of: Biotechniques. Volume 29:Number 4(2000)
• Page Start: 786
• Page End: 792
• Publication Date: 2000-10
• Subjects: Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/00294st05
• Languages: English
• ISSNs: 0736-6205

Abstract

We have developed a rapid, microplateformat plasmid isolation procedure to purify sequencing-grade DNA templates for high-throughput DNA sequencing operations. A modified lysozyme/boiling method is used to produce a plasmid-containing supernatant that is then purified by iron bead capture. After binding, the beads are pelleted in a magnetic field, washed and the DNA eluted in water. The method yields up to 10 µg plasmid DNA from a 1-mL overnight culture in a deep-well microplate. The procedure is suitable for large-scale experiments, amenable to automation and does not require expensive reagents or equipment. The entire protocol can be completed in as little as 2 h, and one technician with a 96-well pipetting station can process up to 48 plates per day. This protocol is ideal for any highthroughput operation in which template quantity, quality and reproducibility are of primary importance.