Team:Paris Bettencourt/Protocols/Heat shock transformation for yeast

Transformation yeast Protocol
  • After growth, determine the titer of the yeast culture by using spectrophotometer : pipette 10µL of cells into 1mL of wtaer in spectrophotometer cuvette and measure the OD at 600nm.
  • Add 2.5x108 cells to 50mL of 2X YPD in a culture flask.
  • Incubate the flask in a shaking incubator at 30°C until the cell culture is at least 2x107 cells.mL-1
  • Denature 1mL of carrier DNA at 99°C for 5min and chill immediately in ice.
  • Harvest the yeast cells by centrifugation at 3,000g for 5min.
  • Resuspend the pellet in 25mL of sterile water and centrifuge at 3,000g for 5min at 20°C. Repeat this wash with sterile water 2 times.
  • Resuspend the last pellet in 1mL of sterile water.
  • Transfer the cell suspension to a 1.5mL microcentrifuge tube.
  • Centrifuge for 30s at 13,000g and discard the supernatant.
  • Resuspend the cells in 1mL of sterile water and pipette 100µL samples into 1.5mL microcentrifuge tubes, one for each transformation.
  • For each transformation :
    • 240µL of PEG 3350 (50% (w/v))
    • 36µL of LiAc 1.0M
    • 50µL of single-stranded carrier DNA (2.0mg.mL-1)
    • 6µL of PCR product
    • 28µL of water DNAse Free
  • Place the tubes at 42°C for 40min.
  • Centrifuge the tubes at 13,000g for 30s in a microcentrifuge tube and remove the supernatant.
  • Pipette 1mL of YPD liquid medium into the transformation tube, and vortex mix to resuspend pellet.
  • Incubate 3h at 30°C to ensure good antibiotic expression.
  • Plate 2, 20 and 200µL of the cell suspension onto YPD medium with 200µm.mL-1 antibiotic G418.
  • Incubate the plates at 30°C for 3 days.