Preparation of Saccharomyces cerevisiae receptor cells

  1. Pick a fresh Saccharomyces cerevisiae EBY100 monoclonal from YPD plates into 10 mL YPD liquid medium and incubate at 30°C, 250 rpm overnight.
  2. Determine the OD600 value of the overnight culture to be between 3.0 and 5.0.
  3. Dilute 10 mL of YPD overnight culture to an OD600 value of 0.2~0.4.
  4. Continue to incubate in a shaker at 28~30°C for 3~6 hr to reach an OD600 value of 0.6~1.0.
  5. Centrifuge the yeast cells for 5 min at 1500 g at room temperature and discard the supernatant.
  6. Wash yeast cells with 10 mL of washing solution, followed by centrifugation at 1500 g for 5 min at room temperature to collect the cells, and discard the supernatant.
  7. Resuspend yeast cells with 1 mL TE/LiAc in 50 μL portions per tube.

Make LB and YEDP media:

  1. Take a small amount of yeast strain Y2H frozen stock and incubate it in line on YPDA plate medium, inverted at 30°C.
  2. Pick a single colony of yeast in a 2.0 mL centrifuge tube, add 1 ml of YPD liquid medium and shake vigorously to disperse the clumps.
  3. Transfer 1:100 ratio to the appropriate amount of YPD liquid medium, incubate at 30°C, 220 rpm for 18-24 h, until stable, OD600>1.5.
  4. Transfer the culture to YPD liquid medium at a ratio of 1:4 and incubate at 30°C, 220 rpm for 3 h until OD600=0.2-0.3.
  5. Transfer the yeast culture to a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 1 min at room temperature, and discard the supernatant.
  6. Add 1 mL of TE buffer, re-suspend the precipitate, centrifuge at 12000 rpm for 30 s, and discard the supernatant.
  7. Add 0.1 mL of TE-LiAc solution and re-suspend the precipitate.

Transformation of Saccharomyces cerevisiae cells

  1. Take 50 µL of receptor cells, add 2 µL of each plasmid to be transformed, and mix well
  2. Add 500 µL of transformation solution (PEG/LiAc, dimethyl sulfoxide), bounce on the wall of the tube and mix well.
  3. Incubate in a shaker at 30°C for 1 hour, and mix by bouncing the wall of the tube at 15-minute intervals.
  4. Add 1 mL of YPD culture solution and incubate for 1 hour at 30℃ in a shaker.
  5. Centrifuge at 3500 g for 5 minutes, leave the sediment and discard the supernatant.
  6. Re-suspend the precipitate with 150 μL TE, apply the corresponding SD plate and place in a 37℃ incubator for 30-60 minutes until the surface liquid has penetrated into the medium, then invert the plate and place in a 37℃ incubator overnight.

Verification of grown yeast cells using PCR and electrophoresis

  1. Jump out the cultured yeast from the YPD plate and place in YPD solution.
  2. Use a shaking incubator at 37℃ for 2-3 hours to grow more yeast cells.
  3. PCR after taking out of the incubator.
  4. 1 uL DNA, 5 uL Mix, 2 uL forward primer, 2 uL reverse primer, 10 uL ddH2O.
  5. Place on a shaker and beat well.
  6. Centrifuge in a centrifuge for 1s.
  7. put into thermal cycler for DNA replication.
  8. Prepare solution: TE buffer, and agarose gel.
  9. ddH2O is added to the TE buffer solution, and the solution is then used as a solvent to melt the agar.
  10. After adding agar, microwave for one minute.
  11. Load the PCR mixture into the agarose gel for electrophoresis: total left to right Marker, FAS1, FAS2, ACC1.
  12. Finally check the results to determine if the desired yeast cells are cultured.