User:Anthony Salvagno/Notebook/Research/Yeast Genomic DNA Prep

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This procedure should describe all/most of the steps involved in extracting yeast genomic DNA. Protocols for creating certain chemical solutions can be found in my Notebook on the days I created them (see Table of Contents on the main Research page).



  1. Grow yeast culture (I started with wt w303a)
  2. Inoculate started culture in liquid YPD.
    • Store in 30C for > 4hrs
  3. Measure OD 600nm
  4. Dilute to OD=0.0005 in 25mL YPD
    • Store in 30C overnight
  5. Measure OD (hopefully will be around .5)
  6. Spin 10 ODs of cells
  7. Resuspend pellet in 5mL of sheroplasting buffer (SB) with 50mM 2-ME and 150uL lyticase
  8. Incubate on ice
    • monitor OD of 1/4 dilutions in 1% SDS (let sit in SDS for 2 min before measuring)
    • OD should drop from .5 to < .1 over about 30 min
  9. Add 5mL Proteinase K Buffer (PK; to be made fresh)
  10. Incubate for 30 min at 65C
  11. Add 2mL 5M KOAc
    • ice for 10 min
  12. spin 10 min at 10Krpm in SS34 tube
  13. collect supernatant in fresh SS34 tube (~10mL)
  14. add 30mL EtOH
  15. precipitate ~20C for 60 min
    • this is a stable state, so it could be a good stopping point. You would be able to end here for the day and go home.
  16. spin 10 min
  17. resuspend pellets in 500uL TE
    • transfer to 2mL eppi
  18. add RNaseA to
    • incubate 37C for 30 min
  19. extract with Phenol/Chloroform
  20. EtOH precipitate DNA (~400uL plus 1.2mL EtOH)
  21. spin 10 min in microfuge
  22. dissolve DNA in 50-100uL TE
  23. measure DNA concentration on nanodrop

Possible changes

  • In step 7 (adding lytocase), in my first trial we needed to add an extra 100uL of lyticase because the reaction was taking too long. This worked well.
    • Total reaction time took 105 min after adding more lyticase at the 60 min point.