A simplified protocol for the semi-large scale recovery of plasmids from Escherichia coli grown on agar plates

Masahiro Sato, Eri Akasaka, Issei Saitoh, Masato Ohtsuka, Shingo Nakamura, Takayuki Sakurai, Satoshi Watanabe

Animal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Ibaraki, Japan.
Department of Organ Regeneration, Graduate School of Medicine, Shinshu University, Nagano, Japan.
Department of Pediatric Dentistry, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
Department of Surgery II, National Defense Medical College, Saitama, Japan.
Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa, Japan.
Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, Japan.

DOI: 10.4236/jbise.2012.57051                

Abstract

Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100-200 ml) has been widely used for the acquisition of relatively large amounts of plasmid DNA (50-300 μg). However, this method requires an expensive high-speed centrifugation apparatus to precipitate E. coli before lysis, which is both laborious and time-consuming. Here, we demonstrate a method for agar plate-based cultivation of bacteria that does not employ a high-speed centrifugation apparatus. This procedure proves to be simple and reproducible, yielding an average of 82 μg of plasmid DNA per experiment. It may therefore be valuable for cloning/transfection experiments under limited financial backgrounds.

Keywords

Agar Plate; DNA Purification; E. coli; Gene Engineering; Plasmid

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	DNA Purification in Promega Home Page (http://www.promega.com/resources/product-guides-and-selectors/protocols-and-applications-guide/dna-purification/)
[2]	QIAGEN Plasmid Purification System in Qiagen Home Page (http://www.ebiotrade.com/buyf/productsf/qiagen/QIAGEN_plasmid_purification_system.htm)
[3]	Sambrook, J., Fritsche, E. and Maniatis, T. (1989) (Ed.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press.
[4]	Sato, M., Ishikawa, A. and Kimura, M. (2002) Direct injection of foreign DNA into mouse testis as a possible in vivo gene transfer system via epididymal spermatozoa. Molecular Reproduction and Development, 61, 49-56.
[5]	Niwa, H., Yamamura, K. and Miyazaki, J. (1991) Efficient selection for high-expression transformants with a novel eukaryotic vector. Gene, 108, 193-200.
[6]	van Ooyen, A., van den Berg, J., Mantei, N. and Weissmann, C. (1979) Comparison of total sequence of a cloned rabbit beta-globin gene and its flanking regions with a homologous mouse sequence. Science, 206, 337-344.