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Yeast Transfection (Small Scale)

Materials

  • YPDA Media 50 mL in a 250 mL flask and 300 mL in a 1L flask
  • TE/LiAc prepare fresh by combining 150 uL 10X TE, 150 uL 1M LiAc and 1.2 mL water
  • PEG/LiAc prepare fresh by adding 8 mL 50% PEG-3350 to 1 mL 10X TE and 1 mL 1M LiAc in a 15 mL falcon tube
  • Herring testes carrier DNA. Boil 20min then place on ice before use

Protocol

  1. Grow 50mL overnight culture of yeast in YPDA or SD. Add several colonies to 1mL vortexing to break up clumps then pouring into 50 mL media in a 250 mL Erlenmeyer flask. Grow at 30C
  2. ransfer overnight culture to 300 mL YPDA to a OD600 of 0.2-0.3
  3. Incubate 3h at 30C to an OD of 0.4-0.6
  4. Place Herring DNA on heating block at 95C for 20min then immediately on ice before use.
  5. Pour cells into 50 mL Falcon tubes and centrifuge 1000g for 5min
  6. Discard supernatant and resuspend in 50 mL sterile TE or water
  7. Centrifuge 1000g for 5min
  8. Discard supernatant and resuspend in 1.5 mL of TE/LiAc. This is enough for about 14 transformations
  9. For each cotransformation combine 0.2 ug Bait (pGBT) and 0.1 ug Prey (pGADGH) with 0.1 mg Herring DNA (10 uL)
  10. dd 100 uL yeast cells and mix by vortexing
  11. Add 0.6 mL PEG/LiAc solution and vortex to mix
  12. Incubate at 30C for 30min with shaking
  13. Add 70 uL DMSO
  14. Heat shock for 15min at 42C swirling occasionally to mix
  15. Chill on ice for 1-2 min
  16. Centrifuge at high speed for 5 s to pellet cells
  17. Resuspend in 0.5mL TE
  18. Plate 200 uL for cotransformation or 100 uL for single transformation onto appropriate selective media
  19. Allow cells to grow for 3-5 days on selective media.
Last modified on 14 December 2009, at 15:02