Volume 12 Part 1 Article 16: Isolation of DNA from Agrocybe aegerita for the Construction of a Genomic Library in Escherichia coli

Volume 12 Part 1 Article 16
Year 1989
Title: Isolation of DNA from Agrocybe aegerita for the Construction of a Genomic Library in Escherichia coli
Authors: T. Noel and J. Labarere

Abstract:

Molecular biology techniques and gene tjransfer systons were recently introduced in Basidiomycetes, notably in Schizophyllum commune (Dons et al. 1984; Ullrich et al. 1985; Mulder G.H., Wessels J.G.H. 1986; Munoz-Rivaz et al. 1986), and in Coprinus cinereus (Binninger et al. 1987).

Our aim was to study the genome of the edible Basidiorycete Agrocybe aegerita using gene cloning. Thus, we report here on early results obtained in the steps leading to the construction of a gencmic library of A. aegerita. We investigated several methods for extracting DNA, problems arising from ligation between digested DNA and plasmid vector, and transformation efficiencies of an Escherichia coli strain.

Many DNA isolation techniques have been described in eucaryotic cells. Results vary regarding yield, size and purit y of DI^ depending on the organism studied, the methods used, and the preparation of tissues or cells prior to DNA isolation.

Recombinant DNA techniques require high molecular weight DNA highly purified. We investigated six methods for DNA isolation and purification adapted to freeze-dried mycelium, liquid nitrogen frozen mycelium and protoplasts of A. aegerita. Methods yielding large fragments of DNA were retained in a first time. DNA was then partially digested with the restriction endonuclease Sau3A and the restriction fragments recoiibinated with the dephosphorylated BamHl digested pBR322. The efficiency of ligation was checked visually by an agarose gel electrophoresis, and was assessed by using the ligation mixtures to transform E. coli strain BJ5183.

This study resulted in the determination of the optimal conditions for preparing DNA and for its further use in the construction of a gencmic library of A. aegerita.

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