A modified alkaline lysis method for the preparation of highly purified plasmid DNA from Escherichia coli.
We have developed a very efficient and rapid method for the preparation on a small or large scale of highly purified plasmid DNA from Escherichia coli. The procedure consists of five steps: (1) cell lysis by NaOH-SDS, (2) precipitation of cell lysate with 2 M potassium acetate-1 M acetic acid, (3) precipitation of the resulting supernatant with isopropanol, (4) treatment of the precipitate with RNase, and (5) a second isopropanol precipitation. The new procedure yields a plasmid DNA that is more than 90% in the supercoiled form and virtually free from proteins, RNA, and chromosomal DNA. We have thoroughly tested the method in the preparation of several thousand samples of different plasmids from various E. coli strains. We found that it consistently produced samples of plasmid DNA suitable for all routine uses such as restriction analysis, sequencing, and preparation of DNA probes for cloning and hybridization experiments. Moreover, plasmids purified by this procedure could fully replace plasmids purified on CsCl gradients for more demanding tasks such as the in vitro synthesis of RNA probes by phage RNA polymerases, the generation of deletion mutants with exonuclease III, and the transfection of mammalian cells by the calcium phosphate coprecipitation method, as tested on human fibroblasts and on CV-1 cells.
Feliciello
I
I
CEINGE, Dipartimento di Biochimica e Biotecnologie Mediche, IIa Facoltà di Medicina e Chirurgia, Università di Napoli, Italy.
Chinali
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G
D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D000882:Haplorhini; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000502:physiology; D008722:Methods; D010957:Plasmids / Q000235:genetics*; D012967:Sodium Dodecyl Sulfate; D012972:Sodium Hydroxide; D014162:Transfection
D004269:DNA, Bacterial; D012967:Sodium Dodecyl Sulfate; D012972:Sodium Hydroxide