Rapid large-scale purification of plasmid DNA by medium or low pressure gel filtration. Application: construction of thermoamplifiable expression vectors

Autor: Tuyen Vo-Quang, A. Donny Strosberg, Dominique Buffard, P. Alexandre Kaminski, Dominique Vidal, Yves Malpiece
Přispěvatelé: Immunocytochimie, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)
Rok vydání: 1985
Předmět:
Zdroj: Bioscience Reports
Bioscience Reports, 1985, 5 (2), pp.101-111. ⟨10.1007/BF01117056⟩
ISSN: 1573-4935
0144-8463
Popis: International audience; 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.
Databáze: OpenAIRE
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