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).

THIS PAGE IS UNDER CONSTRUCTION

Procedure

  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.
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