This paper describes a new method of lasmid DNA purification which is fast and reliable enough for most purposes in recombinant DNA technology. The present method does not require the use of toxic chemicals such as phenol or ethidium bromide, costly ultra-centrifugation procedures or other processes which can modify the supercoiled structure of the plasmids, such as adsorption on glass fiber. This method is based on the principle of gel filtration chromatography, at low pressure (1 bar) or medium pressure (between 5 and 10 bars), using Sephacryl S1000 or Superose 6B. It permits recovery oI plasmids: (I) in preparative quantities (from 300 gg to 4 mg), (II) exempt from RNA, DNA and protein contamination, and (III) suitable for various common genetic engineering procedures immediately after purification. To test the reliability of the technique as well as the degree of purilication, the plasmids were used to construct thermoampliIiable vectors, carrying the tacUV5 promoter and the 5′ end of the β -gallactosidase gone with a single EcoRl site in each of the three possible translational phases. This set of vectors is designed for the expression of foreign genes as hybrid proteins in Escherichia coli.
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February 01 1985
Rapid large-scale purification of plasmid DNA by medium or low pressure gel filtration. Application: construction of thermoamplifiable expression vectors
Tuyen Vo-Quang;
Tuyen Vo-Quang
1Laboratoire d'Immunocytochimie, Institut Pasteur, Paris 75015, France
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Yves Malpiece;
Yves Malpiece
2Laboratoire G3, Institut Pasteur, Paris 75015, France
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Dominique Buffard;
Dominique Buffard
3Laboratoire d'Immunologie Moléculaire, Institut Jacques Monod, Université Paris VII, 75251 Paris Cedex 05, France
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P. Alexandre Kaminski;
P. Alexandre Kaminski
3Laboratoire d'Immunologie Moléculaire, Institut Jacques Monod, Université Paris VII, 75251 Paris Cedex 05, France
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Dominique Vidal;
Dominique Vidal
2Laboratoire G3, Institut Pasteur, Paris 75015, France
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A. Donny Strosberg
A. Donny Strosberg
3Laboratoire d'Immunologie Moléculaire, Institut Jacques Monod, Université Paris VII, 75251 Paris Cedex 05, France
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Publisher: Portland Press Ltd
Received:
January 16 1985
Online ISSN: 1573-4935
Print ISSN: 0144-8463
© 1985 The Biochemical Society
1985
Biosci Rep (1985) 5 (2): 101–111.
Article history
Received:
January 16 1985
Citation
Tuyen Vo-Quang, Yves Malpiece, Dominique Buffard, P. Alexandre Kaminski, Dominique Vidal, A. Donny Strosberg; Rapid large-scale purification of plasmid DNA by medium or low pressure gel filtration. Application: construction of thermoamplifiable expression vectors. Biosci Rep 1 February 1985; 5 (2): 101–111. doi: https://doi.org/10.1007/BF01117056
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