title: Rapid DNA, RNA and protein extraction protocols optimized for slow continuously growing yeast cultures.
abstract: Conventional extraction protocols for yeast have been developed for relatively rapid-growing low cell density cultures of laboratory strains and often do not have the integrity for frequent sampling of cultures. Therefore, these protocols are usually inefficient for cultures under slow growth conditions or of non-laboratory strains. We have developed a combined mechanical and chemical disruption procedure using vigorous bead-beating that can consistently disrupt yeast cells (> 95%), irrespective of cell cycle and metabolic state. Using this disruption technique coupled with quenching, we have developed DNA, RNA and protein extraction protocols that are optimized for a large number of samples from slow-growing high-density industrial yeast cultures. Additionally, sample volume, the use of expensive reagents/enzymes, handling times and incubations were minimized. We have tested the reproducibility of our methods using triplicate/time-series extractions and compared these with commonly used protocols or commercially available kits. Moreover, we utilized a simple flow-cytometric approach to estimate the mitochondrial DNA copy number. Based on the results, our methods have shown higher reproducibility, yield and quality.
authors: Institute for Advanced Biosciences, Keio University, Yamagata, Japan.
authors: Kalesh
authors: K
authors: Sasidharan
authors: Cornelia
authors: C
authors: Amariei
authors: Masaru
authors: M
authors: Tomita
authors: Douglas B
authors: DB
authors: Murray
mesh_terms: D004271:DNA, Fungal / Q000302:isolation & purification*; D004272:DNA, Mitochondrial / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods; D005656:Fungal Proteins / Q000302:isolation & purification*; D018628:Gene Dosage; D007218:Industrial Microbiology / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D012331:RNA, Fungal / Q000302:isolation & purification*; D015203:Reproducibility of Results; D015003:Yeasts / Q000737:chemistry* / Q000235:genetics*
chemical_list: D004271:DNA, Fungal; D004272:DNA, Mitochondrial; D005656:Fungal Proteins; D012331:RNA, Fungal