Purification of plasmid and high molecular mass DNA using PEG-salt two-phase extraction.
A method for the rapid preparation of DNA is described. The method utilizes a polymer (polyethylene glycol) and salt solution to form a two-phase system. A crude source of DNA is added to a phase-forming mixture, it is mixed and phase separation occurs. Under the appropriate conditions, the nucleic acids remain in the lower (salt-rich) phase, while the proteins, cellular debris and other constituents are in the upper phase (polymer-rich) or are precipitated at the interphase region. Incorporation of protein denaturants (detergents and chaotropes) stop the action of liberated nucleases in the sample. The nucleic acids are obtained in an intact state and in a form suitable for further manipulation, as shown by gel electrophoresis and DNA restriction digestion. This method describes the conditions of the two-phase systems that are important for the separation of nucleic acids and proteins. The important phase-forming conditions shown in this paper are pH, polymer molecular weight and concentration, salt type and concentration and the addition of detergents and chaotropic agents. With the use of these extraction conditions, proteins can be moved selectively from the lower to the upper phase. The paper describes a method for DNA isolation that is rapid, simple and economical.
Cole
K D
KD
Center for Chemical Technology, National Institutes of Standards and Technology, Boulder, CO 80303.
D004247:DNA / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005821:Genetic Techniques; D010957:Plasmids; D011092:Polyethylene Glycols
D011092:Polyethylene Glycols; D004247:DNA