Isolation of yeast genomic DNA Sample Clauses

Isolation of yeast genomic DNA. Single colonies were used to inoculate 10 mL YPD cultures, which were grown with shaking (200 rpm) overnight at 30°C. The following morning, 1 mL of cells was pelleted via centrifugation at 3500 rpm for 2 minutes in an FA-45-24-11 rotor (Eppendorf). The supernatant was removed and the cells were resuspended in 200 μL of lysis buffer (100 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, and 0.1% SDS) and mixed with sterile glass beads. The mixture was vortexed at maximum for 1 minute, mixed with 200 μL of phenol, and then spun down for 2 minutes at maximum speed in a FA-45-24-11 rotor (Eppendorf). The aqueous layer was removed and extracted a second time with an equal volume of phenol (as above). Two volumes of ethanol was then added to the aqueous layer, which was mixed and left on dry ice to precipitate the DNA. The DNA was pelleted by spinning for 15 minutes (maximum speed) at 4°C in a FA-45-24-11 rotor (Eppendorf). The supernatant was removed and the pellet was left to dry at room temperature. Once dry, the pellet was resuspended in TE pH 8.0 and left to dissolve overnight at 4°C.