How to make yeast competent cells and yeast cell transformation?

How to Make and Transform Competent Yeast Cells

Competence is a special physiological state of a cell in which a cell can take up exogenous DNA from the surrounding environment. Using lithium acetate and polyethylene glycol (PEG), yeast cells can be made competent for transformation and subsequent downstream applications.

In this method, lithium acetate changes cell permeability to allow uptake of foreign DNA. To protect yeast cell membrane from the high concentration of lithium acetate and allow the plasmid to contact the cell membrane more tightly for an efficient transformation, PEG is added to the transformation solution. The squid sperm monomer DNA (ssDNA), a short linear single-stranded DNA, can also be added to the transformation cocktail to prevent DNA degradation by yeast DNase and also to promote the absorption of foreign DNA.

Protocol for Making Yeast Competent Cells

1. Take out frozen yeast cells from -80°C freezer and streak on a YPD agar plate. Incubate in a stationary incubator at 30°C for 2 days.

2. Pick a single colony and inoculate a 250 mL flask containing 50 mL of YPD medium. Incubate overnight at -30°C for 12~16 hours or overnight on a shaking incubator with enough aeration (200~300 r/min).

3. Add about 1 mL of the culture into a 250 mL flask containing 50 mL of YPD medium, and incubate at 37°C for 4~5 hours (200~300 r/min).

4. When the 600 nm OD value reaches 0.35~0.4, transfer the culture into a 50 mL centrifuge tube and centrifuge at 3000~4000 rpm for 5 minutes.

5. Discard the supernatant and add 30 mL of pre-chilled ddH₂O to the centrifuge tube. Re-suspend cells and centrifuge at 3000 rpm for 10 minutes.

6. Discard the supernatant and add 30 mL of 0.1M LiAc to the centrifuge tube. Re-suspend cells, and centrifuge at 3000 rpm for 5 minutes.

7. Repeat step 6.

8. Discard the supernatant and add 500µL of 0.1M LiAc to the centrifuge tube. Re-suspend cells and dispense 100 µL of the pellet into five 1.5 mL Eppendorf tubes.

9. Proceed with transformation or store cells for future use by re-suspending cells with equal volumes of 0.1M LiAc and 10% glycerol. Dispense 100 µL of cells into five 1.5 mL Eppendorf tubes and store at -80°C freezer.

Protocol for Transforming Yeast Competent Cells

1. Prepare the transformation solution as follows (total volume: 900 µL)

PEG 3350 (50% w/v) . . . . . . . . . . . . . . .  . . . . . . .. 660 µL

ssDNA (10 mg/mL) . ..  . . . . . .. . . . . . .  . . . . . . .. 35 µL

LiAc (1.0M) . . . . . . . . . . . . . . . .. . . . . . .  . . . . . . . 90 µL

Plasmid DNA (0.1 µg) plus sterile water . . . . . .  .. .115 µL

2. Thaw frozen yeast competent cells at room temperature. If using freshly-made competent cells, go to step 3.

3. Centrifuge cells at 4000 rpm for 3 minutes to remove the supernatant. Add 100 µL of 0.1M LiAc to re-suspend cells.

4. Add 900µL transformation solution to competent cells, mix gently and incubate at room temperature for about 20 minutes. Include a negative control (i.e. without plasmid DNA) tube.

5. Transfer the tube to 42°C water bath and hold for 40 minutes.

6. Centrifuge at 4000 rpm for 2 minutes. Discard the supernatant and then add 100 of 0.1M CaCI₂ to re-suspend cells.

7. Plate 100~300 µL of cells on YNB + MA plates.

8. Incubate plates at 30°C for 2~4 days for colonies to grow.

Tips from the MolecularCloud™ Team

1. Make sure you slowly freeze recently-made competent yeast cells. Place yeast competent cell tubes in a Styrofoam or cardboard box for storing at -80°C freezer.

2. Before preparing the transformation solution, boil the ssDNA for 10 minutes and then place it on ice to open the double-stranded DNA. This ensures that the ssDNA exists in a single-stranded form in the transformation solution.

3. For the preparation of yeast competent cells, it is not necessary to maintain the temperature at 4°C, maintain the room temperature.