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Factors affecting chemical‐based purification of DNA from Saccharomyces cerevisiae
Author(s) -
Lee Christopher K.,
Araki Naoko,
Sowersby Drew S.,
Lewis L. Kevin
Publication year - 2012
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.1918
Subject(s) - lysis , biology , alkaline lysis , yeast , dna , dna extraction , saccharomyces cerevisiae , sodium acetate , lysis buffer , chromatography , yield (engineering) , restriction enzyme , sodium , tris , sodium hydroxide , extraction (chemistry) , cell disruption , microbiology and biotechnology , polymerase chain reaction , biochemistry , plasmid , chemistry , gene , dna vaccination , materials science , organic chemistry , metallurgy
ABSTRACT Extraction of high molecular weight chromosomal DNA from yeast cells is a procedure that is performed frequently for experiments involving polymerase chain reaction (PCR), Southern blotting and other DNA analysis techniques. We have investigated several parameters affecting DNA yield and quality, using a simple chemical‐based purification procedure that was modelled on alkaline lysis methods developed for bacterial cells. The three major steps of the procedure, cell lysis, protein removal and DNA precipitation, were optimized by testing the impacts of several chemicals, including sodium dodecyl sulphate (SDS), sodium hydroxide, Tris buffer, sodium acetate and potassium acetate. Other parameters, such as the effect of elevated temperatures on cell lysis, were also investigated. A rapid, optimized protocol was derived for the purification of DNA from small cell cultures that can be readily digested with restriction enzymes and used as a template for PCR. Average yield was calculated to be approximately 1.7 µg DNA/10 8 cells, which is similar to the theoretical maximum amount obtainable from haploid yeast cells. Copyright © 2011 John Wiley & Sons, Ltd.