Raught Lab 2008
Yeast Genomic DNA prep.
** It is important to work quickly, but carefully,
and to keep samples on ice as much as possible **
1. Grow O/N culture (5ml YPD)
2. Pellet cells at 3000rpm for 4 minutes
3. Resuspend pellet in 0.5ml sorbitol solution
4. Add 5ul 20T Zymolyase (3mg/ml) and 12ul of 1.14M β–mercaptoethanol (1.14M is the standard concentration of commercially bought β–mercaptoethanol). Vortex gently
5. Incubate the tubes for 1 hour @ 37°C
6. Centrifuge at 3000rpm, discard supernatant. Resuspend pellet in 0.5ml Tris/EDTA solution and move to fresh eppendorf
7. Add 25ul of 20% SDS and invert tubes to mix
8. Incubate for 20min @ 65°C to lyse the cells (should get a viscous solution)
9. Add 200ul of 5M KAc (potassium acetate), invert to mix, set on ice for 30-45min
10. Centrifuge @ 12000rpm for 3min at room temperature
11. Collect supernatant into fresh eppendorf (~0.7ml of sup.). Add 1ml of 100% EtOH* to sup. And mix by inverting.
12. Centrifuge @ 12000rpm for 2min @ 4°C. Wash pellet with 1ml of 70% EtOH*.
13. Centrifuge @ 12000rpm for 2min @ 4°C. Remove as much of the sup. as possible. Use a fine tip pipette to remove residue. Air dry tube/pellet for 10-15min
14. Resuspend pellet in 50ul of H2O or TE buffer.
*EtOH should be ice cold. Generally an aliquot is kept in the -20°C freezer for this purpose.
Raught Lab 2008
Sorbitol Solution:
Final / Product / Stock / Amount0.88M / Sorbitol / 1M / 45ml
0.98M / Tris pH 8.0 / 1M / 5ml
0.098M / EDTA / 0.5M / 1ml
Tris/EDTA Solution:
Final / Product / Stock / Amount10mM / Tris pH 8.0 / 1M / 100ul
1mM / EDTA / 0.5M / 20ul
Sterile water / to 10ml