Editing Yeast Genomic DNA (section)

== <br>Quick Yeast Genomic DNA Prep ==

<br>1) Grow 10 ml yeast cultures to saturation in YEPD.<br>2) Pour the culture into a 15 ml conical and spin at 3000 rpm for 3 min. Dump sup. Resuspend in 500 µl distilled water.<br>3) Transfer to microfuge tube. Spin at 13000 rpm for 1 min. Dump sup. Vortex briefly to resuspend the pellet in the residual liquid.<br>4) Add 200 µl detergent lysis buffer, 200 µl phenol chloroform (at 4°C, take bottom layer), and ~300 mg glass beads (1 scoop).<br>5) Vortex 3-4 minutes on multihead vortex.<br>6) Add 200 µl TE pH 8. Spin at 13000 rpm for 5 minutes. Transfer aqueous layer (top) to a fresh tube.<br>7) Add 1 ml of ice cold 100% EtOH. Mix by inversion.<br>8) Spin at 13000 rpm for 2 minutes at 4°C. Dump sup. Resuspend in 400 µl TE pH 8 and 3 µl RNase.<br>9) Incubate 5 minutes at 37°C.<br>10) Add 10 µl 4 M ammonium acetate plus 1 ml of ice cold 100% EtOH. Mix by inversion<br>11) Spin at 13000 rpm for 2 minutes at 4°C. Dump sup. Air-dry pellet.<br>12) Resuspend in 50 µl TE.<br>13) Store at -20°C.

=== <br>Materials: ===

<br>-Saturated culture<br>-Detergent lysis buffer:<br>2% Triton X 100<br>1% SDS<br>100 mM NaCl<br>10 mM Tris-Cl pH 8<br>1 mM EDTA<br>-PCI: Phenol, chloroform, IAA (25:24:1)<br>-acid-washed glass beads<br>-TE pH 8<br>-10 mg/ml boiled RNase<br>-4 M ammonium acetate<br><br>