Identification and eradication of a denatured DNA isolated during alkaline lysis-based plasmid purification procedures.
Many plasmid isolation procedures use strongly alkaline conditions in the initial stages to facilitate lysis of the host bacteria. We demonstrate that such procedures can give rise to a minor but significantly altered form of plasmid. After electrophoresis and uv transillumination of ethidium bromide-stained agarose gels we and others have noticed a faint band migrating near to the major fluorescent product, covalently closed circular plasmid DNA. This faint band is resistant to cleavage by restriction endonucleases which have recognition sites in the parent plasmid. We were able to show that the contaminating band is able to transform competent Escherichia coli cells and that normal double-stranded plasmids were isolated from such transformants. We were able to selectively hydrolyze the contaminating band using T5 exonuclease which is a 5'-nuclease with a single-strand specific endonuclease activity. Plasmid preparations carried out under nonalkaline conditions failed to produce the contaminant band. We suggest methods for purifying plasmid DNA which remove the denatured band and could improve cloning efficiencies where the largest recombinant libraries are required.
Sayers
J R
JR
University of Sheffield, Section of Molecular Medicine, Krebs Institute, Royal Hallamshire Hospital, United Kingdom. j.r.sayers@sheffield.ac.uk
Evans
D
D
Thomson
J B
JB
D004274:DNA, Recombinant / Q000737:chemistry* / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D006863:Hydrogen-Ion Concentration; D009691:Nucleic Acid Denaturation; D010957:Plasmids
D004274:DNA, Recombinant