Editing Yeast Genomic DNA

== <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> 



== Genomic DNA Extraction S. cerevisiae and S. pombe<br> ==

=== Protocol: ===

<br>1. Grow a 10ml overnight culture, spot onto YPD to check markers<br>2. Pellet cells, decant supernatant and resuspend in 0.5ml water<br>3. Transfer to a 1.7ml micro-centrifuge tube<br>4. Pellet 5 seconds and decant supernatant<br>5. Briefly vortex pellet to resuspend in remaining medium<br>6. Add: 0.2ml Smash and Grab Solution<br> 0.2ml Phenol-Chloroform<br> 0.3grams Acid-washed glass beads<br>7. Vortex for 2-5 minutes using the multi-tube holder or 10 minutes on multi-tube vortexer in the<br> cold room<br>8. Add 0.2ml of TE to each tube<br>9. Spin 5 minutes in centrifuge<br>10. Transfer aqeous phase to new tube<br>11. Add 1.0ml EtOH and mix to ethanol precipitate<br>12. Spin down 5 minutes, decant supernatant<br>13. Resuspend pellet in 0.4ml TE<br>14. Add 30μg RNase and incubate for 5 minutes at 370<br>15. Add 10μl 4M ammonium acetate and 1.0ml EtOH<br>16. Ethanol precipitate on ice for 5 minutes<br>17. Spin down 10 minutes<br>18. Optional wash with 0.5ml 70% EtOH<br>19. Air dry pellet and resuspend in 100μl TE<br>Entered by Diana Libuda on 12/6/2007

=== <br>Smash and Grab Solution (100mL) ===

<br>10 mL 10% SDS (1%)<br>2 mL 100x Triton-X100 (2%)<br>2 mL 5M NaCL (100mM)<br>1 mL 1M Tris pH 8.0 (10mM)<br> (1mM)<br>Mix in beaker, adding the detergents while the solution is mixing. Filter sterilize.

[[Category:Protocols]]