Transformation of Yeast
(download the PDF here)
(This protocol is taken from Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, 2005 Edition)
- Inoculate 5 ml of liquid YPD or 10 ml of SC and incubate with shaking overnight at 30C.
- Count overnight culture and inoculate 50ml of YPD to a cell density of 5 X 106 cells/ml of culture.
- Incubate the culture at 30C on a shaker at 200 rpm until it is at 2 x 107 cell/ml. This typically takes 3-5 hours. This culture will give sufficient cells for ten transformations.
Notes:
- It is important to allow the cells to complete at least two divisions.
- Transformation efficiency remains constant for three to four divisions.
- Harvest the culture in a sterile 50-ml centrifuge tube at 3000g (2500 rpm) for 5 minutes.
- Pour off the medium, resuspend the cells in 25 ml of sterile H2O, and centrifuge again.
- Pour off the H2O, resuspend cells in 1.0 ml of 100mM lithium acetate (LiAc), and transfer the suspension to a sterile 1.5-ml microfuge tube.
- Pellet the cells at top speed for 5 seconds and remove the LiAc with a micropipette.
- Resuspend the cells to a final volume of 500 µl (2 x 109 cells/ml), which is about 400 ml of 100 mM LiAc.
Note: If the cell titer of the culture is grater than 2 x 107 cell/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, decrease the amount of LiAc.
- Boil a 1.0-ml sample of single-stranded carrier DNA for 5 minutes and quickly chill in ice water.
Note: It is not necessary or desirable to boil the carrier DNA every time. Keep a small aliquot in your freezer box and boil after three or four freeze/thaws.
- Vortex the cell suspension and pipette 50-µl samples into labeled microfuge tubes. Pellet the cells and remove the LiAc with a micropipette.
- The basic “transformation mix” consists of the following ingredients; carefully add them in the order listed:
240 µl of PEG (50% w/v)
36 µl of 1.0M LiAc
25 µl of single-stranded carrier DNA (2.0 mg/ml)
50 µL of H2O and plasmid DNA (0/1-10mg)
Note. The order is important here. The PEG, which shields the cells from the detrimental effects of the high concentration of LiAc, should go in first.
- Vortex each tube vigorously until the cell pellet has been completely mixed. This usually takes 1 minute.
- Incubate for 30 minutes at 30C.
- Heat shock for 20-25 minutes at 42C
Note: The optimum time can vary for different yeast strains. Test this if you need high efficiency from your transformations.
- Microfuge at 6000-8000 rpm for 15 seconds and remove the transformation mix with micropipette.
- Pipette 0.2-1.0 ml of sterile H2O into each tube and resuspend the pellet by pipetting it up and down gently.
- Plate from 200-µl aliquots of the transformation mix onto selective plates.