genome-sequencing of saccharomyces cerevisiae
After washing in ddH2O, cells were resuspended in 300 L breaking buffer [2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (pH 8.0)]. After addition of 300L acid-washed glass beads (BioSpec, Cat. No.11079105, 0.5 mm dia.) and 300 L phenol/chloroform/ isoamylol (25:24:1), cells were ruptured by vigorous vortexing for 5 min. Then 200 L ddH2O was added into the tube, followed with slight shaking and centrifuged at 12000 rpm for 10 min. The aqueous upper phase was transferred to a 1.5 ml Eppendorf tube, followed with adding 1 ml of absolute ethanol and mixing the contents by inversion. After centrifugation at 12000 rpm for 10 min, the pellet was dried in vacuum pump and dissolved in 400 L TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). To digest the RNA, 3 L of a 10 mg/mL RNaseA solution was added into the tubes, incubated at 37 for 120 min. To precipitate the genomic DNA, 1 mL of absolute ethanol and 10 L of 5M ammonium acetate were added. The pellet was collected by centrifugation at 12000 rpm for 10 min, dried in vacuum pump and resuspended in 100 L of TE buffer as samples.