A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformaion of Escherichia coli

doi:10.1016/0378-1119(87)90131-4

Gene

Volume 57, Issues 2–3, 1987, Pages 267-272

https://doi.org/10.1016/0378-1119(87)90131-4 Get rights and content

Abstract

A procedure for the rapid isolation of DNA from the yeast Saccharomyces cerevisiae is described. To release plasmid DNA for the transformation of Escherichia coli, cells are subjected to vortex mixing in the presence of acid-washed glass beads, Triton X-100, sodium dodecyl sulfate, phenol and chloroform. Centrifugation of this mixture separates the DNA from cellular debris. E. coli can be efficiently transformed with plasmid present in the aqueous layer without further purification of the plasmid DNA. This procedure also releases chromosomal DNA. Following two ethanol precipitations, the chromosomal DNA can be digested by restriction endonucleases and analysed by Southern blot analysis.

References (15)

D. Botstein et al.
Sterile host yeast (SHY): a eukaryotic system of biological containment for recombinant DNA experiments
Gene
(1979)
D.R. Cryer et al.
Isolation of yeast DNA
Methods Cell. Biol.
(1975)
C. Holm et al.
A rapid, efficient method for isolating DNA from yeast
Gene
(1986)
R. Losson et al.
Plasmids carrying the yeast OMP decarboxylase structural and regulatory genes: transcription regulation in a foreign environment
Cell
(1983)
G.S. Roeder et al.
DNA rearrangements associated with a transposable element in yeast
Cell
(1980)
E.M. Southern
Detection of specific sequences among DNA fragments separated by gel electrophoresis
J. Mol. Biol.
(1975)
F. Winston et al.
Eviction and transplacement of mutant genes in yeast
Methods Enzymol.
(1983)

There are more references available in the full text version of this article.

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Short communicationA ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformaion of Escherichia coli

Abstract

Gene

Methods Cell. Biol.

Gene

Cell

Cell

J. Mol. Biol.

Methods Enzymol.

Short communication
A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformaion of Escherichia coli