Yeast chemical transformations - glasgowlab/home GitHub Wiki

Original method in this paper: Yeast LiAc transformations
This works great with EBY100 / S. cerevisiae.

Prepping for the yeast transformation

  1. Start a 5 ml cell culture by transferring 5 ml of yeast media (e.g. YPD) into a culture tube and inoculating the tube with a single colony.
    Note: If yeast cells are grown in media without antibiotics, use careful sterile technique to not contaminate the cultures. Also always grow yeast in a flask to avoid the cells sinking into the bottom of tubes.
  2. Grow the yeast cell cultures shaking overnight at 30°C.
  3. The next morning, measure the O.D. of the cell culture(s) 600 nm and dilute the cell culture(s) down to a starting O.D. of 0.25. Dilute the cell culture(s) with the yeast media that was used to grow the cell cultures the night before.
  4. Grow the diluted cell cultures back up to an O.D. of 0.7 (this usually takes about 4 hours).

The transformation

  1. Transfer 50 ml of the cell cultures into conical tube(s) and centrifuge the tube(s) for 3 minutes at 3000 rpm. Be sure to use all of the cell cultures!
  2. Pipette the supernatant out of the tube(s) and discard it.
  3. Re-suspend the pellet(s) in the conical tube(s) with 800 µl of H2O.
  4. Transfer the re-suspended pellet(s) into eppendorf tube(s) and centrifuge the tube(s) for 5 minutes at 3000 rpm. 5.Discard the supernatant and estimate the volume of pellet formed in a single tube. Match that pellet volume to the volume of H20 needed to re-suspend the pellet(s).
  5. Pipette 50 µl of the re-suspended cells into new tube(s). This transfer contains 25 µl of cells and 25 µl of water.
  6. Spin the tube(s) down again for 1 minute and 30 seconds at 3000 rcf. When centrifuge is complete, discard the liquid. Then, to each tube, add ingredients:
    • 36 uL of 1M LiAc
    • 10 uL ssDNA carrier (2 mg/ml), prepared by boiling for 5 min then placing on ice
    • 1 uL of plasmid (or 15 uL of PCR product) (you want ~1 ug)
    • 59 uL of H2O (or 44 uL if using PCR product)
      NOTE: LiAc "loosens" the cells' membranes to allow the DNA into the cells. ssDNA aids the transforming DNA into the cells so transformation can take place within the cells.
  7. Next, re-suspend the pellet of cells gently.
  8. After, pipette 240 µl of 50% PEG 3350 in the tube(s) and VORTEX the tubes. Incubate 30 min at RT.
  9. Add 50 uL DMSO after incubation is complete.
  10. Incubate the tubes in a 42C water bath for 40 minutes to heat shock.
    Note: PEG is very viscous and thus, if it is added first, it becomes very difficult to re-suspend the cells evenly throughout the mixture, making transformation of the cells very difficult. PEG helps the cells stay in the mixture throughout, while minimizing gravity's effect.
  11. Once the last step is finished, spin the tubes down once more for 3 minutes at 3000 rpm.
  12. Dump the supernatant and re-suspend pellet(s) in 350 µl of yeast media.
  13. Incubate cells at 30C for 4h to rescue from heat shock (this is critical).
  14. Then, plate the cells on desired selective media and grow in a 30C incubator for approximately 3 days (or more).
    NOTE: Do not need to plate all of the cells, a quarter or a sixth is plenty.

After 3 days have elapsed, proceed to yeast colony PCR.