Drummond:Yeast Genomic DNA Prep Protocol

Protocol

• Inoculate a 3 mL YPD culture with a single yeast colony and grow to saturation (overnight) at 30C with shaking or rotation.

• Transfer 500 uL or 750 uL of cells to a microcentrifuge tube and pellet at top speed for 10 sec. Dump supernatant and wash cells with 0.5ml distilled water, and pellet at top speed for 10 sec.

• Remove wash using a vacuum trap being careful to not disturb the pellet.

• Add the following:

0.2ml DNA Extraction Buffer 0.2ml phenol:chloroform:isoamyl alcohol (25:24:21) 0.3g glass beads (use tube measurement)

• Vortex for 3 min. Add 0.2ml TE (pH 8), and transfer entire contents to a phase-lock tube.

• Centrifuge for 5 min at top speed, and transfer the aqueous top phase to a clean microcentrifuge tube.

• Add 1ml of 100% EtOH. Mix by inversion.

• Centrifuge for 2 min at top speed. Remove supernatant using a vacuum trap being careful to not disturb the pellet.

• Dissolve pellet in: 0.4ml of TE.

• Add 5µl of 10mg/ml RNase A and mix by inversion. Incubate for 30 min at 37°C in water bath. Add 10µl of 4M ammonium acetate and 1ml of 100% EtOH. Mix by inversion.

• Centrifuge for 2 min. Remove supernatant using a vacuum trap being careful to not disturb the pellet.

• Air dry pellet or dry in vacuum oven for 10 min and resuspend in 50µl of EB. Check concentration with the Nanodrop spectrophotometer.

• Optional: check the quality of the genomic DNA prep by electrophoresis on a 0.8% agarose gel.