96-well Yeast Genomic Prep Protocol

  • Option #1:  Start 200 μL YPD cultures, in 96 well 500 μL round bottom assay plate, and grow cultures on plate shaker overnight at 30°C
    1. Spin down cultures for 1 minute at 4000 rpm in a table top centrifuge
    2. Remove supernatant
    3. Add cell suspension solution and proceed with the protocol
  • Option #2:  Place a colony with a clean pipet tip directly into the Cell Suspension solution (see below) and proceed with the protocol

  1. For each sample mix 2 μL of Lytic Enzyme Solution  to 150 μL of Cell Suspension Solution
  2. Pipet 150 μL of Cell Suspension Solution + Lytic Enzyme Solution  into each well of the 96-wel plates (500 μL round bottom assay plates)
  3. Pick a colony with a clean pipet tip and mix directly into the Cell Suspension Solution + Lytic Enzyme Solution  and vortex briefly
  4. Incubate at 37°C for 20 minutes
  5. Centrifuge for 1 minute at 4000 rpm on (table top centrifuge)
  6. Vacuum off supernatant
  7. Add 150 μL of the Cell Lysis Solution  and resuspend cell pellet by vortexing briefly
  8. Incubate at 65°C (dryer) for 10 minutes
  9. Add 50 μL of the Protein Precipitation Solution  and mix by vortexing briefly
  10. Incubate at 4°C for 10 minutes
  11. Spin down at 4000 rpm for 3 minutes
  12. Transfer 100 μL of the supernatant to a clean plate
  13. Add 100 μL of isopropanol to each well and mix by vortexing
  14. Incubate 15+ minutes at 4°C
  15. Spin down at 4000 rpm for 5 minutes
  16. Vacuum off the supernatant
  17. Add 150 μL of 70% EtOH to DNA pellet
  18. Incubate 15+ minutes at 4°C
  19. Spin down at 4000 rpm for 5 minutes
  20. Vacuum off the supernatant
  21. Resuspend DNA in 20-50 μL TE

Solution Recipes

  • Cell Suspension Solution:
    • 1 M Sorbitol, 0.1 M EDTA
  • Lytic Enzyme Solution (store at 4°C)
    • 7.5 mg/mL Zymolase in Cell Suspension Solution
  • Cell Lysis Solution
    • 50 mM Tris-Cl (pH 7.4), 20 mM EDTA, 1% SDS
  • Protein Precipitation Solution
    • 5 M Potassium Acetate