High efficiency method for LiAc/SS-DNA/PEG transformation of yeast

Gietz R.D. and Shiestl R.H. (1995). Transforming yeast with DNA. Methods in Molecular and Cellular Biology 5, 255-269.

Day 1

1. Inoculate 2-5 mls of liquid YPD or 10 ml SC
   Incubate with shaking overnight at 30°C

Day 2

1. Count o/n culture and inoculate 50 mls of warm YPD to a cell density of 5 x 10⁶/ml culture.
   ! Dilute overnight YPD or SC cultures 10^-1 or more in water.
2. Incubate the culture at 30°C on a shaker at 200 rpm until its equivalent to 2 x 10⁷ cells/ml. This will take 3 to 5 hours.
   This culture will give sufficient cells for 10 transformations.
   ! It is important to allow the cells to complete at least two divisions.
   ! Transformation efficiency (transformants/ µg plasmid/10⁸ cells) remains constant for 3 to 4 celldivisions.
   ! Alternatively, cultures can be prepared as described in protocol 2.2.4.1. (steps 1-3 of day 1)
3. Harvest the culture in a sterile 50 ml centrifuge tube at 3000 x g (5000 rpm) for 5 min.
4. Pour off the medium, resuspend the cells in 25 ml of sterile water and centrifuge again.
5. Pour off the water, resuspend the cells in 1.0 ml 100 mM LiAc and transfer the suspension to a 1.5 ml microfuge tube.
6. Pellet the cells at top speed for 15 sec and remove the LiAc with a micropipette.
7. Resuspend the cells to a final volume of 500 µl (2 x 10⁹ cells/ml) (i.e., about 400µl
   of 100 mM LiAc)
   ! If the cell titer of the culture is greater than 2 x 10⁷/ cells ml the volume of the LiAc should be increased to maintain the titer of this suspension at 2 x 10⁹ cells/ml. If the titer of the culture is less than 2 x 10⁷/ cells ml then decrease the amount of LiAc.
8. Boil a 1.0 ml sample of SS-DNA for 5 min. and quickly chill in ice water.
   ! It is not necessary or desirable to boil the carrier DNA every time. Keep a small aliquot in your own freezer box and boil after 3-4 freeze-thaws. But keep on ice when out
9. Vortex the cell suspension and pipette 50 µl samples into labelled microcentrifuge tubes. Pellet the cells and remove the LiAc with a micropipette.
10. The basic "transformation mix" consists of:

240 µl PEG (50% w/v)
36 µl 1.0 M. LiAc
10 µl SS-DNA (10 mg/ml)
X µl Plasmid DNA (0.1 - 10 µg)
74-X µl Sterile ddH2O
360 µl TOTAL


Carefully add these ingredients in the order listed.
! The order is important here! The PEG should go in first, which shields the cells from the detrimental effects of the high concentration of LiAc. One can also premix the ingredients except for the plasmid DNA then add 355µl of TRAFO mix on top of the cell pellet. Then add the 5 µl of plasmid DNA and mix. Take care to deliver the correct volume as the Trafo mix is viscous.

11. Vortex each tube vigorously until the cell pellet has been completely mixed. Usually takes about 1 min.
12. Incubate at 30°C for 30 min.
13. Heat shock in a water bath at 42°C for 30 min.
    ! The optimum time can vary for different yeast strains.
14. Microfuge at 6000 rpm for 15 sec
    Remove the transformation mix with a micropipette.
15. Pipette 1.0 ml of sterile water into each tube and resuspend the pellet by pipetting it up and down gently.
    ! We like to be a gentle as possible at this step if high efficiency is important. Excessive washing washes away transformants!!!!
16. Plate from 2 to 200 µl of the transformation mix onto SC-minus plates. If plating less than 200 µl deliver into a pool of not more than a final volume of 200 µl of sterile water on the plate.
    When spreading yeast inoculum onto the plate gently distribute the fluid completely with a sterile glass rod with a minimum of strokes. Allow the fluid to be taken up by the plate prior to incubation.
17. Incubate the SC minus plates for 2 - 4 days to recover transformants.

Polyethylene glycol (PEG 50% w/v)
The polyethylene glycol (PEG), MW 3350 (Sigma P3640) is made up to 50% (w/v) with dd water and filter sterilized.
! For optimal transformation efficiencies, care must be taken to ensure that the PEG solution is at the proper concentration. In addition, it is important to store the PEG in a tightly capped container to prevent evaporation of water and a subsequent increase in PEG concentration. Small variations above or below the PEG concentration optimum in the transformation reaction, which is 33% (w/v), can reduce the production of transformants.
1.Place 50 gm of polyethylene glycol, MW 3350 (Sigma) in a 150 ml glass beaker and add 35 mls of ddH20.
2.Stir with a magnetic stirring bar until dissolved. This will take about 30 min.
3.Transfer all of the liquid to a 100 ml graduated cylinder. Rinse the beaker with a small amount of distilled water, add this to the graduated cylinder containing the PEG solution, and bring the volume to exactly 100 mls. Mix well by inversion.
4. Autoclave or filter sterilize using a 0.45 µm filter unit (Nalgene), and store in a securely capped bottle.