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.