Abstract
A procedure is described for the isolation and purification ofE. coli plasmid DNA by polyethylene glycol precipitation. The method is rapid, simple, inexpensive and amenable to both small and large scale manipulation. This procedure involves lysis of bacterial cells by treatment with pronase in sodium dodecyl sulfate, removal of chromosomal DNA by centrifugation, precipitation of residual nucleic acids with polyethylene glycol and removal of RNA by precipitation with LiCl. Plasmid DNA purified as described is pure enough for restriction endonuclease analysis, for use as a vector for the cloning of cDNA or synthetic DNA, or for use as a template in anE. coli transcription-translation cell-free system.
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Abbreviations
- PEG:
-
polyethylene glycol
- TE buffer:
-
10 mM Tris-HCl pH 7.5, 1 mM EDTA
- SDS:
-
sodium dodecyl sulfate
- tet:
-
tetracycline
- amp:
-
ampicillin
- kan:
-
kanamycin
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Pulleyblank, D., Michalak, M., Laurent Daisley, S. et al. A method for the purification ofE. coli plasmid DNA by homogeneous lysis and polyethylene glycol precipitation. Mol Biol Rep 9, 191–195 (1983). https://doi.org/10.1007/BF00775367
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DOI: https://doi.org/10.1007/BF00775367