title: Enhancing yields of low and single copy number plasmid DNAs from Escherichia coli cells.
abstract: Many plasmids used for gene cloning and heterologous protein expression in Escherichia coli cells are low copy number or single copy number plasmids. The extraction of these types of plasmids from small bacterial cell cultures produces low DNA yields. In this study, we have quantitated yields of low copy and single copy number plasmid DNAs after growth of cells in four widely used broths (SB, SOC, TB, and 2xYT) and compared results to those obtained with LB, the most common E. coli cell growth medium. TB (terrific broth) consistently generated the greatest amount of plasmid DNA, in agreement with its ability to produce higher cell titers. The superiority of TB was primarily due to its high levels of yeast extract (24g/L) and was independent of glycerol, a unique component of this broth. Interestingly, simply preparing LB with similarly high levels of yeast extract (LB24 broth) resulted in plasmid yields that were equivalent to those of TB. By contrast, increasing ampicillin concentration to enhance plasmid retention did not improve plasmid DNA recovery. These experiments demonstrate that yields of low and single copy number plasmid DNAs from minipreps can be strongly enhanced using simple and inexpensive media.
authors.affiliation: Chemistry and Biochemistry, Texas State University, 601 University Drive, San Marcos, TX 78666, United States.
authors.forename: Whitney N
authors.initials: WN
authors.lastname: Wood
authors.affiliation: Chemistry and Biochemistry, Texas State University, 601 University Drive, San Marcos, TX 78666, United States.
authors.forename: Kyle D
authors.initials: KD
authors.lastname: Smith
authors.affiliation: Chemistry and Biochemistry, Texas State University, 601 University Drive, San Marcos, TX 78666, United States.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Ream
authors.affiliation: Chemistry and Biochemistry, Texas State University, 601 University Drive, San Marcos, TX 78666, United States. Electronic address: LL18@txstate.edu.
authors.forename: L Kevin
authors.initials: LK
authors.lastname: Lewis
mesh_terms: D001431:Bacteriological Techniques; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D056915:DNA Copy Number Variations; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D010957:Plasmids
chemical_list: D003470:Culture Media; D004269:DNA, Bacterial
keywords: Copy number; DNA purification; LB; Miniprep; Plasmid; TB