Saccharomyces cerevisiae Transformation

Preparing yeast competent cell

  1. Fresh culture in 3mL YPD at 28C overnight.
  2. Inoculate 0.5mL fresh culture into 50mL YPD, OD 0.4, and incubate at 28C with shaking for 3hr.
  3. Centrifuge for 5min at 4000rpm, 4C, and resuspend in 25mL 1X TE, 0.1M.
  4. Centrifuge for 5min at 4000rpm, 4C, and resuspend in 0.5mL sterile 1X TE / 0.1M LiAc.

Transformation

  1. Make a mixture according to the composition below.
  2. Add salmon sperm DNA 10uL.
  3. Add DNA 1-2ug, 10uL.
  4. Add yeast competent cell 100uL.
  5. Add PEG/LiAc 600uL.
  6. Incubate at 30C for 30min.
  7. Add DMSO 70uL and invert to resuspend.
  8. Heat shock for 15min at 42C.
  9. Chill tube on ice for 3min and centrifuge at 8000rpm for 3min at 4C.
  10. Discard supernatant.
  11. Resuspend cell in 100uL TDW or TE buffer and spread onto plate.
  12. Incubate plate for 2-3 days.
  13. For LiAc/TE, combine 10X TE 5mL, 10X LiAc 5mL, and water 40mL.