Process for obtaining plasmid dna by means of an...

C - Chemistry – Metallurgy – 12 – N

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C12N 15/10 (2006.01)

Patent

CA 2527216

The invention relates to a method for the isolation of plasmid-DNA from biomasses, by means of an aqueous biphasic system, comprising a polymer and a salt component, characterised in that the resuspension of the applied biomass, the alkaline lysates of the biomass, the neutralisation of the alkaline lysate component and the separation of the plasmid-DNA from the contaminants (such as cell remains, RNA and gDNA, for example) is carried out in a single reaction vessel (one-pot method). According to the invention, the above is achieved, whereby the neutralisation of the alkaline lysate component is carried out in one and the same container by the addition of potassium phosphate and hence one component of the aqueous biphasic system is already present and the second component of the aqueous biphasic system is a PEG with a molecular weight of calculated average from about 600 g/mol to 1000 g/mol, however, said second component is preferably formed by a mixture of PEG 600 and PEG 1000.

L'invention concerne un procédé pour isoler un ADN plasmidique de biomasse au moyen d'un système biphasique aqueux pourvu d'un constituant polymère et d'un constituant salin. Ledit procédé se caractérise en ce que la remise en suspension de la biomasse utilisée, la lyse alcaline de la biomasse, la neutralisation du lysat alcalin et la séparation de l'ADN plasmidique des contaminants (tels que les débris cellulaires, l'ARN, l'ADN génomique) sont mises en oeuvre dans un réacteur unique (procédé monotope). A cet effet, la neutralisation du lysat alcalin est effectuée dans un seul et même récipient par l'ajout de phosphate de potassium, un constituant du système biphasique aqueux étant ainsi déjà présent, et le second constituant de ce système biphasique aqueux est un PEG d'un poids moléculaire moyen en nombre compris entre environ 600 g/mol et 1 000 g/mol, mais de préférence formé à partir d'un mélange de PEG 600 et de PEG 1000.

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