Identification and Eradication of a Denatured DNA Isolated during Alkaline Lysis-Based Plasmid Purification Procedures
✍ Scribed by Jon R. Sayers; Debbie Evans; James B. Thomson
- Publisher
- Elsevier Science
- Year
- 1996
- Tongue
- English
- Weight
- 108 KB
- Volume
- 241
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
tion and precipitation as originally described by Birn-Many plasmid isolation procedures use strongly alboim and Doly (6). Recent discussions on the BIONET kaline conditions in the initial stages to facilitate lynewsgroup have revealed that several groups as well sis of the host bacteria. We demonstrate that such as our own have noticed an apparent contaminant in procedures can give rise to a minor but significantly alkaline lysis-prepared plasmids (7). The contaminant altered form of plasmid. After electrophoresis and uv is visualized upon analysis by agarose gel electrophoretransillumination of ethidium bromide-stained agarsis. The band appears as one of variable but low fluoose gels we and others have noticed a faint band mirescent intensity upon ethidium bromide staining. The grating near to the major fluorescent product, covafluorescent intensity of the contaminating band is usulently closed circular plasmid DNA. This faint band ally weak relative to the major desired plasmid prodis resistant to cleavage by restriction endonucleases uct, circular covalently closed DNA (cccDNA). 2 The miwhich have recognition sites in the parent plasmid. nor product appears to be recalcitrant to digestion by We were able to show that the contaminating band is restriction endonucleases even under conditions which able to transform competent Escherichia coli cells completely cleave the normal plasmid DNA. The con-