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genomic dna isolation from yeast for 96-well plate

Yeast Genomic DNAs for 96-well plate

  1. Grow cells on plate of KIWI 2-3 days @ 28°C.
  2. Then scrape cells off and resuspend in 7.5/10 ml (col./rows) of 1 M sorbitol. Split into 1.4 ml aliquots and store @ -80°C.
  3. Thaw on tube and pellet 1 min top sped @ RT.
  4. Decant and resuspend pellet in 1 ml of SCE with 1% ß-Mercaptoethanol (1 µl/ml) and 1 mg/ml Zymolase (make fresh); use 500 µl first pipet up and down and then add another 500 µl; vortex.
  5. Incubate @ 37°C for 40 min; pellet 2 min @ RT.
  6. Resuspend (pipet up and down; vortex) in 750 µl of fresh lysis solution [0.5% SDS, 100 mM Tris pH 8.8, 50 mM EDTA pH 8.8]
  7. Heat @ 70°C for 15 min (vortex in between if not 100% dissolved) and put on ice.
  8. Add 75 µl of 5.0 M KOAc, vortex and leave on ice 30 min.
  9. Pellet 7 min top speed @ RT and decant SN into new tube and add 700 µl of isopropanol, mix and vortex well, pellet 2 min top speed @ RT.
  10. Rinse pellet twice with 70% EtOH and drain.
  11. Resuspend in 0.5 ml of T5E [50mM Tris pH 8.0, 10 mM EDTA pH 8.0].
  12. Add 330 µl of 7.4 M NH4OAc and mix.
  13. Pellet 5 min top speed @ RT.
  14. Transfer the SN to new tube and add 0.7 ml of isopropanol, mix and vortex well, and pellet 2 min top speed @ RT.
  15. Rinse pellet twice with 70% EtOH and drain.
  16. Resuspend in 0.5 ml of T5E and add 50 µl of 3 M NH4OAc and mix.
  17. Add 0.5 ml of isopropanol, vortex, pellet 2 min top speed @ RT.
  18. Rinse pellet twice with 70% EtOH. Then add 100 µl of 96% EtOH, pellet for 1 min top speed @ RT and remove liquid with p200 pipette. Dry pellet and resuspend in 0.5 ml of TE [~0.5 µg/µl]


SCE

  • 91.1 g Sorbitol
  • 14.7 g NaCitrate
  • 11.2 g EDTA

bring to 500 ml with water and pH to 7.0 with 0.1 N NaOH and filter sterilize.


Lysis solution

  • 0.5% SDS
  • 100 mM Tris pH 8.8
  • 50 mM EDTA pH8.8


T5E

  • 50 mM Tris pH 8.0
  • 10 mM EDTA pH 8.0