Yeast Genomic (Chromosomal DNA) Prep
1. Grow cultures overnight in 5-6 mL of YPD at 30°C regardless of plasmids or selection.
2. Spin tubes in clinical centrifuge for 3 minutes at between ¾ and ½ speed.
3. Pour off the YPD media and save pellet!
4. Resuspend into 500 uL of “Genomic Solution #1” (1 M sorbitol, 0.1 M Na2EDTA pH 7.5; filter
sterilized; higher pH still works). Vortex briefly to mix too.
5. Add 5 uL of Zymolyase solution (store at 4°C, 25 mg/mL in 50% glycerol; vortex stock
solution to ensure mixed).
6. Place on rotator at 37°C for at least 1 hour (can go slightly longer too).
7. Spin for 11,000 rpm for 1 minute.
8. Pipette off the solution and keep pellet.
9. Resuspend (gently, the pellet will be “goopy”) in 500 uL of “Genomic Solution #2” (50 mL TrisCl pH 7.4, 20 mM Na2EDTA; filter sterilized). Vortex for 20 seconds to ensure mixed.
10. Add 50 uL of 10% SDS solution.
11. Vortex for 10 seconds to mix. Solution may become “bubbly”.
12. Incubate at 65°C for 30 minutes in heating block.
13. Add 200 uL of 5M KOAc (potassium acetate) and mix by inversion 10 times.
14. Place on ice for 1 hour (can be 45 minutes or longer here).
15. Spin at max speed for 5 minutes.
16. Pour into a fresh Eppendorf tube (labeled) and contains 550 uL of isopropanol (2-propanol).
17. Mix by inversion and WATCH as the DNA will precipitate out (forms long stringy strands).
18. Let sit 30 seconds and DNA will sink to the bottom.
19. Spin for 1 minute at max speed.
20. Pour off the isopropanol and keep pellet (which will be slightly cloudy and very small).
21. Spin a second time for 30 seconds at 8,000 rpms.
22. Pipette off the remaining isopropanol and wait 5 minutes for the rest to evaporate.
23. Add 300 uL of TE solution (10 mM Tris-Cl pH 7.4 and 1 mM Na2EDTA; filter sterilized).
24. Incubate for 10-15 minutes allowing the pellet to soak in the TE.
25. Use a pipette to carefully resuspend the pellet. Pipette TE onto the pellet and “displace” it off
the bottom of the tube, repeat. DO NOT get the pellet stuck inside the pipette tip. The pellet
should be nearly clear (and can be difficult to see). This “pre” resuspension step allows more
buffer to hit the pellet surface and speeds up resuspension.
26. Incubate at room temp another 10-15 minutes.
27. Fully resuspend the pellet now (pipette up and down and the pellet should dissolve and fully
disappear).
28. Add 1.5 uL of RNAaseA solution (stored at -20°C freezer at 10 mg/mL solution in water;
boiled for 10 minutes initially, then freeze and store indefinitely).
29. Incubate at 37°C on rotator (or in rack) for 30 minutes.
30. Add 30 uL of 3 M sodium acetate (autoclaved) and vortex briefly.
31. Add 200 uL of isopropanol and gently invert to precipitate the DNA (WATCH DNA form solid
as you invert tube. This solid should be much smaller than the last isopropanol precipitation).
32. Allow pellet to fall to bottom of tube.
33. Spin at max for 1 minute.
34. Pour off isopropanol solution.
35. Spin at 8,000 rpm for 30 seconds.
36. Pipette off isopropanol solution. Allow pellet to air dry for 2-5 minutes.
37. Add 80-100 uL of TE solution. Allow incubation for 10-15 minutes at room temp.
38. Carefully resuspend pellet into TE by pipetting up and down.
39. Can store at -20°C freezer. Use genomic preps for PCR and use 2.0-2.3 uL per 50 uL PCR
reaction.