Yeast Transformation

Lithium Acetate Transformation (yields a high efficiency)

COMPETENCY:

  1. Inoculate 2-5 ml of liquid YPD and incubate 30oC on shaker overnight.

  2. measure optical density of culture and inoculate 50 ml of warm YPD to a cell density of 5E6 cells/ml culture, O.D.(600) = 0.25.

  3. Incubate at 30oC on shaker at 200 rpm about 3-5 hours, to a cell density of 2E7 cells/ml, O.D.(600) = 1.0. This culture will give sufficient cells for 10 transformations.

  4. Harvest cells at 5,000 rpm for 5 minutes in a 50 ml centrifuge tube.

  5. Pour off supernatant, resuspend cells in 25 ml sterile water, and spin again.

  6. Pour off supernatant, resuspend cells in 1.0 ml 100 mM LiAc and transfer to a 1.5 ml eppendorf tube.

  7. Pellet the cells at 7,000 rpm for 15 seconds and remove LiAc with a micropipette.

  8. Resuspend the cells to a final volume of 1 ml (2E9 cells/lml) with about 700 ul of 100 mM LiAc. (Adjust volume of LiAc to achieve a titer of 2E9 cells/ml.)

  9. Boil a 1.0 ml sample of 2 mg/ml SS-DNA for 5 minutes and quickly chill in ice water. (We have a stock of salmon sperm DNA prepared for this protocol). It is not necessary or desirable to boil the carrier DNA every time. Keep a small aliquot in the freezer and boil after 3-4 freeze-thaws.

  10. Vortex the cell suspension and pipette 100 ul samples into 10 labeled microfuge tubes. Pellet the cells and remove the LiAc with a micropipette.

TRANSFORMATION:
  1. The basic transformation mix consists of:
    • 240 ul PEG (50% w/v)
    • 36 ul 1.0 M LiAc
    • 50 ul SS-DNA (2.0 mg/ml)
    • 50 ul H2O and plasmid DNA (0.1-10 ug)
    Add these ingredients in the order listed, leaving the cells as a pellet.

  2. Vortex or pipette each tube vigorously until the pellet is completely mixed. DO NOT VORTEX CELLS AGAIN AFTER THIS STEP.

  3. Incubate at 30oC for 30 minutes.

  4. Heat shock in H2O bath at 42oC for 20-25 minutes.

  5. Microfuge at 4,000 rpm for 8 seconds and remove the transformation mix with a micropipette.

  6. Pipette 0.4 ml of sterile distilled H2O into each tube and resuspend the pellet gently.

  7. Plate on selective media and incubate for 2-4 days to recover transformants.


PEG-TEL Transformation (yields a pretty decent efficiency)

COMPETENCY:

  1. Inoculate a single colony into 50 ml YPD.

  2. Grow on shaker to O.D.(600) = 1.0 (O.D.(600) between 0.6 and 1.8 is fine) at 30oC overnight.

  3. Spin down cells in table top centrifuge at 2,000 rpm.

  4. Wash cells in 5 ml distilled H2O.

  5. Spin down cells and resuspend in 500 ul distilled H2O.

  6. Use for transformation.

TRANSFORMATION:
  1. Pellet 50 ul competent cells in a 1.5 ml eppendorf tube by pulsing up to top speed in the microcentrifuge.

  2. Remove supernatant.

  3. Add 276 ul 43% PEG-LiAC, resuspend thoroughly.

  4. Add 50 ul salmon sperm DNA (usually 2 mg/ml).

  5. Add 50 ul DNA to suspension. Typically 1 ul of miniprep DNA for uncut plasmids, or 20 ul of digested plasmid (bring up to 50 ul with distilled H2O).

  6. Heat shock at 42oC for 40 minutes. Plate on appropriate selective plates.


43% PEG-TEL buffer

TO MAKE 115 ml: 

  • 100 ml 50% PEG-3350 stock solution
  • 15 ml 1 M LiAC stock solution