A nonalkaline method for isolating sequencing-ready plasmids.
We describe a simple method of isolating plasmid DNA directly from Escherichia coli culture medium by addition of lithium acetate and Sodium dodecyl sulphate, followed by centrifugation and alcohol precipitation. The plasmid is sufficiently pure that it can be used in many enzyme-based reactions, including DNA sequencing and restriction analysis. Chromosomal DNA contamination is significantly reduced by pretreatment of the culture with DNase I, suggesting that much of the contaminant is associated with permeable dead cells. Chromosomal DNA contaminant can also be selectively denatured without damage to the supercoiled plasmid by alkaline denaturation in an arginine buffer or heat treatment in the presence of urea or N,N-dimethylformamide.
Paul
Bonnie
B
Department of Biology, California State University, Northridge, CA 91330-8303, USA.
Cloninger
Cheri
C
Felton
Marilyn
M
Khachatoorian
Ronik
R
Metzenberg
Stan
S
D000468:Alkalies; D002457:Cell Extracts; D002876:Chromosomes, Bacterial / Q000378:metabolism; D003470:Culture Media; D004269:DNA, Bacterial / Q000302:isolation & purification; D003850:Deoxyribonuclease I / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D010957:Plasmids / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA / Q000379:methods*
D000468:Alkalies; D002457:Cell Extracts; D003470:Culture Media; D004269:DNA, Bacterial; D003850:Deoxyribonuclease I