Yeast Genomic DNA Prep

Linda Hoskins/Hahn lab Aug 18, 1997 (modified from Philippsen, 1991)

Grow 10ml YPD cultures o/n. Figure out cell density; inoculate 30 ml YPD and grow o/n so that cell density is approximately 2 x 108 cells/ml the next morning. Spin down cells in 50 ml sterile conical tubes for 10 min. Centrifugation steps are performed at 4 degrees; all other steps are carried out at rm. temp. unless otherwise specified. Resuspend cells in 10 ml water and spin down cells. Resuspend cells in 3 ml of 0.9 M sorbitol, 0.1 M EDTA, 50 mM DTT, pH 7.5. Add 0.25 mg Zymolyase dissolved in 200 ul 0.9 M sorbitol and incubate with occasional shaking at 37 degrees. Conversion of spheroplasts takes 15-120 min. depending on the strain used, on the growth medium, and on the growth phase. Check for spheroplast formation by mixing 4 ul cells and 4 ul 0.1% SDS on a microscope slide. Formation is complete when 80-90% of the cells are "ghost" cells. Compare to a slide with 4 ul cells and 4 ul sorbitol solution. Spin spheroplasts for 5 min. and carefully discard the supernatant. Resuspend spheroplasts in 3 ml of 50 mM Tris-HCl, 50 mM EDTA, pH 8.0, by slowly and repeatedly drawing the spheroplasts into a pipette. Then mix with 0.3 ml 10% SDS and incubate at 65 degrees for 30 min. Add 1 ml 5 M KOAc, mix, and let sit on ice for 60 min. or longer. The white precipitate that forms consists mainly of insoluble potassium dodecyl sulfate and denatured proteins. Transfer to a 50 ml centrifuge tube and spin at 15,000 rpm for 30 min. in a Sorvall SS34 rotor. Transfer supernatant (about 4 ml) to a 12 ml disposable centrifuge tube. Add 4 ml ice-cold absolute ethanol. On mixing, the nucleic acids (2% DNA and 98% RNA) and some residual proteins with immediately precipitate. Spin at 10,000 rpm for 10 min. and discard the supernatant. Wash with 4 ml 70% ethanol. Spin at 10,000 rpm for 10 min.

Resuspend in 300 ul TE, pH 7.5. Pellet will take a while to dissolve. A 10 min. incubation at 42 degrees can help. May need to let sit O/N in fridge. At this point keep a 3 ul aliquot to compare to prep after Rnase treatment. Add 15 ul 10 mg/ml Dnase-free Rnase and incubate at 37 degrees for 30 min. The stock of Rnase is dissolved in 10 mM sodium acetate, pH 7.0, and kept at �20 degrees. Add 300 ul phenol/chloroform (1:1) and mix by inverting. Spin for 10 min. Transfer supernatant to new tubes. Add 15 ul 3 M NaOAc and 900 ul isopropanol. Ppt. should be immediately visible, if not, put on dry ice for 5 min. Spin for 5-10 min. Wash with 80% EtOH. Air dry pellet. Resuspend in 100-300 ul TE, pH 7.5. Run an aliquot of purified DNA and the aliquots of DNA before RNase treatment on a 0.7% agarose gel. The DNA should be stored at +4 degrees or �70 degrees, not at �20 degrees. Frequent freezing and thawing should be avoided.