title: Identification of RNase-resistant RNAs in Saccharomyces cerevisiae extracts: Separation from chromosomal DNA by selective precipitation.
abstract: High-quality chromosomal DNA is a requirement for many biochemical and molecular biological techniques. To isolate cellular DNA, standard protocols typically lyse cells and separate nucleic acids from other biological molecules using a combination of chemical and physical methods. After a standard chemical-based protocol to isolate chromosomal DNA from Saccharomyces cerevisiae and then treatment with RNase A to degrade RNA, two RNase-resistant bands persisted when analyzed using gel electrophoresis. Interestingly, such resistant bands did not appear in preparations of Escherichia coli bacterial DNA after RNase treatment. Several enzymatic, chemical, and physical methods were employed in an effort to remove the resistant RNAs, including use of multiple RNases and alcohol precipitation, base hydrolysis, and chromatographic methods. These experiments resulted in the development of a new method for isolation of S. cerevisiae chromosomal DNA. This method utilizes selective precipitation of DNA in the presence of a potassium acetate/isopropanol mixture and produces high yields of chromosomal DNA without detectable contaminating RNAs.
authors.affiliation: Chemistry and Biochemistry, Texas State University, 601 University Drive, San Marcos, TX 78666, USA.
authors.forename: Blanca V
authors.initials: BV
authors.lastname: Rodriguez
authors.affiliation: Chemistry and Biochemistry, Texas State University, 601 University Drive, San Marcos, TX 78666, USA.
authors.forename: Eric T
authors.initials: ET
authors.lastname: Malczewskyj
authors.affiliation: Chemistry and Biochemistry, Texas State University, 601 University Drive, San Marcos, TX 78666, USA.
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Cabiya
authors.affiliation: Chemistry and Biochemistry, Texas State University, 601 University Drive, San Marcos, TX 78666, USA.
authors.forename: L Kevin
authors.initials: LK
authors.lastname: Lewis
authors.affiliation: Department of Chemistry, Trinity University, One Trinity Place, San Antonio, TX 78212, USA. Electronic address: cmaeder@trinity.edu.
authors.forename: Corina
authors.initials: C
authors.lastname: Maeder
mesh_terms: D019840:2-Propanol / Q000737:chemistry; D011232:Chemical Precipitation; D002875:Chromosomes / Q000235:genetics*; D004247:DNA / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D019347:Potassium Acetate / Q000737:chemistry; D012313:RNA / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*
chemical_list: D012313:RNA; D004247:DNA; D012260:Ribonucleases; D019347:Potassium Acetate; D019840:2-Propanol
keywords: DNA; Extraction; Precipitation; Purification; RNA; RNase