Yeast Transformation (standard protocol)

Protocol:

  1. Grow 5ml starter culture overnight.
  2. Dilute to OD600 = 0.2 in 40ml of appropriate growth medium.
  3. Grow to OD600 = 0.6-1.0 (Usually 3-6 hours depending on strain).
  4. Collect by centrifuging at 2000 RPM for 5 minutes in 50ml Falcon tube.
  5. Wash once in 10ml dH2O.
  6. Wash once in 5ml TE-LiAc
  7. Resuspend in 1ml TE-LiAc (cells can be used immediately or saved at 4C for up to a week)
  8. Use 100ul cells per transformation:
    1. Add 5ul of 10mg/ml ssDNA.
    2. Add 1-10ul of DNA for transformation.
    3. Add 70ul 10X TE.
    4. Add 560ul 40% PEG-3350.
    5. Add 70ul 1M LiAc.
    6. Vortex.
  9. Incubate at 30C for two hours.
  10. Incubate in 42C water bath for 15 minutes.
  11. Place on ice for two minutes.
  • If using auxotrophic markers (ie, ura, trp etc), cells can be spun down at 6K RPM, resuspended in 300ul dH2O and plated directly (100ul per plate).
  • If using antibiotic (ie kanMX), add transformed cells to 5ml YPD and grow 4-16 hours at 30C. Resuspend cells in 300ul dH2O and place on appropriate antibiotic plates (100ul per plate).

Reagents:

  1. 10mg/ml ssDNA
  2. 10X TE (100mM Tris, pH8;10mM EDTA)
  3. 1M LiAc
  4. TE-LiAc (1X TE;100mM LiAc)
  5. 50% PEG-3350 in dH20 and filter sterilized (Note: Transformation efficiency decreases if the PEG is old, make up fresh every month or two)