Yeast Genomic DNA

Quick Yeast Genomic DNA Prep

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

Materials:

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

Genomic DNA Extraction S. cerevisiae and S. pombe

Protocol:

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

Smash and Grab Solution (100mL)

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