Introduction

High-efficiency transformation of intact yeast cells has been improved dramatically since its original description1,2,3,4,5,6. The yeast transformation method has been recently reviewed7, and the unfamiliar reader should refer to this document concerning application of these techniques. This method can be used to produce frozen competent yeast cells that transform at high efficiency when a single yeast strain is used repeatedly, such as in a yeast two-hybrid screen8,9,10. Yeast cultures re-grown for a least two divisions in rich medium can be used to produce transformation competent cells that can be frozen and stored for later use. In addition, yeast cells carrying a plasmid can also be prepared by growing the cells overnight selectively followed by growth in rich medium for two generations. In most cases, two generations of nonselective growth does not produce dramatic plasmid loss. This technique can be used to grow small or large batches of cells for freezing. One hundred samples of 1 × 108 frozen competent cells will require 500 ml of re-grown culture (1 × 1010 cells). Transformation with frozen competent cells is similar to the high-efficiency method10,11. The cells are thawed, pelleted by centrifugation, mixed with transformation mix and heat shocked.

Materials

Reagents

  • Bacto yeast extract (Fisher Scientific Ltd, cat. no. DF0886-17-0)

  • Bacto peptone (Fisher Scientific Ltd, cat. no. DF0118-17-0)

  • Adenine hemisulfate (Sigma Chemical Co. Ltd, cat. no. A-3159)

  • Bacto Agar (Fisher Scientific Ltd, cat. no. DF0145-17-0)

  • Yeast synthetic drop-out medium supplements (Sigma Chemical Co. Ltd, cat. nos. Y-1376, Y-1751, Y-2001, Y-0750, Y-1876, Y-1501)

  • Sodium hydroxide solution 10 N (Sigma Chemical Co. Ltd, cat. no. 72068)

  • G418 (Sigma Chemical Co. Ltd, cat. no. A-1720)

  • Lithium acetate dihydrate (Sigma Chemical Co. Ltd, cat. no. L-6883)

  • PEG 3350 (Sigma Chemical Co. Ltd, cat. no. P-3640)

  • Salmon sperm DNA (Sigma Chemical Co. Ltd, cat. no. D-1626)

  • Tris–EDTA buffer solution (10 mM Tris–HCl, 1 mM Na2 EDTA, pH 8.0; Sigma Chemical Co. Ltd, cat. no. 93283)

  • Cartridge-purified sterile water

  • Ethanol 99.5% (vol/vol; Sigma Chemical Co. Ltd, cat. no. 459844)

  • Dimethyl sulfoxide (Sigma Chemical Co. Ltd, cat. no. D-2650)

  • Glycerol (Sigma Chemical Co. Ltd, cat. no. G5516)

Equipment

  • 1.5 ml polypropylene microcentrifuge tubes (Fisher Scientific Ltd, cat. no. 05-669-32) are autoclaved to sterilize. These tubes are used for each transformation reaction

  • 12 ml 17 × 100 mm sterile polypropylene culture tube (Fisher Scientific Ltd, cat. no. 05-540-6). These tubes are used for growing 2 ml precultures for inoculation of the transformation culture

  • Nalgene Filter Unit 0.20 μm 500 ml (Fisher Scientific Ltd, cat. no. 50-0020/EMD)

  • 50 ml polypropylene sterile disposable centrifuge tubes, Corning (Fisher Scientific Ltd, cat. no. 05-526B). These tubes are used to collect the grown yeast culture before transformation

  • 100 tube styrofoam rack with lid (Sarstedt Inc., cat. no. 95.64.249). Large box that the Styrofoam rack will fit into (Styrofoam or cardboard) with additional insulation such as Styrofoam chips or newspaper to reduce the air space around the sample box

  • 250 ml baffled Erlenmyer flask, sterile (Fisher Scientific Ltd, cat. no. 10-041-5B) is used to grow the 50 ml YPAD yeast culture for transformation for good aeration. Cap with aluminum foil and autoclave

  • 2 liter baffled Erlenmyer flask, sterile (Fisher Scientific Ltd, cat. no. 10-041-4E) is used to grow the 200 ml 2 × YPAD yeast culture for transformation for good aeration. If you do not use a baffled flask, you must use a larger flask to ensure good aeration. Never put more than 10% flask volume in a non-baffled flask. Cap with aluminum foil and autoclave

  • Hemacytometer (Fisher Scientific Ltd, cat. no. S17040) is used to determine cell numbers in cultures

  • Glass rod bent in a P shape. The rod is made from a glass rod 6 inches in length and bent in a P shape so that the right-hand part of the P is about 1 inch in length. It is used to spread yeast cells onto the plate in a sterile manner

  • Microscope with at least a × 10 objective and a × 10 ocular lens. This is used with the hemacytometer to determine cell numbers in yeast cultures

  • MicroMax Therma/IEC microcentrifuge with an IEC 851 rotor (Fisher Scientific Ltd) is used to pellet yeast cells in 1.5 ml microcentrifuge tubes.

  • IEC Centra IE4 table-top centrifuge with IEC 801 rotor (Fisher Scientific Ltd). This is used to spin down yeast cells from 50 ml cultures

  • Spectrophotometer (Fisher Scientific Ltd, cat. no. S42475P) is used at 600 nm for OD for an alternate method of titering yeast cultures and used with plastic cuvettes (Fisher Scientific Ltd, cat. no. 14-385-938)

  • Precision Low Temperature Incubator Model 815. This is set to 30 °C for growth of liquid and plate cultures. Any incubator with forced air heating will be sufficient

  • New Brunswick Gyrotory Shaker Model G2 (Fisher Scientific Ltd, cat. no. 14-285-729) for growing liquid cultures. This is typically placed in the 30 °C incubator. This is a less costly alternative to a floor model shaking incubator

  • Precision Water Bath Model 181 (Fisher Scientific Ltd, cat. no. 15-474-10). This is used for 42 °C heat shock

  • Inoculating loop (Nichrom wire) (Fisher Scientific Ltd, cat. no. 13-104-5). This is used for streaking and inoculating cultures

  • Sterile flat toothpicks, local grocery store. Place wide end down into a 250 ml beaker and cap with aluminum foil and then autoclave. These can then be used by turning the beaker on its side and removing one at a time

  • Bunsen burner or alcohol burner (Fisher Scientific Ltd, cat. no. S41898). This is used to sterilize inoculation loops and the glass rod used for spreading inoculum onto plates

Reagent setup

  • Yeast The Saccharomyces cerevisiae strains used in a transformation protocol can vary depending on the laboratory and the application. Most S. cerevisiae strains give some level of transformants; however, the efficiency can vary with specific yeast strains. We have used numerous strains for highly efficient transformation. Most strains can be obtained from the American Type Culture Collection. It is important to check the genotype of the strain you will be using for the appropriate markers for the plasmids you want to transform. Transformants are selected using plasmid DNA carrying genes that complement mutations found in the specific yeast strain you are transforming. It is important to ensure that the strain you are using has a corresponding mutation for the plasmid used in the transformation.

  • YPAD medium We have found that good growth correlates with good transformation. We grow the yeast in YPAD medium (1% (w/v) Bacto yeast extract, 2% (w/v) Bacto peptone, adenine hemisulfate 80 mg l−1). The yeast cells to be transformed are usually re-grown for two generations in liquid 2 × YPAD medium (2% (w/v) Bacto yeast extract, 4% (w/v) Bacto peptone, 4% (w/v) glucose, adenine hemisulfate 80 mg l−1).

  • Selection medium Synthetic complete drop-out (SC) medium can be purchased from a number of sources (see REAGENTS) as well as mixed according to the protocol of Rose12. The medium is mixed with double-distilled water and then adjusted to pH 5.6 with 1.0 N NaOH and autoclaved. Plates require 18 g l−1 of Bacto Agar.

    Vectors containing the Kan4MX gene require the eukaryotic antibiotic G418 or geneticin. This antibiotic is supplemented into YPD plates at a concentration of 200–300 μg ml−1 depending on the strain being used. The recipe for YPAD listed above, omitting adenine hemisulfate, can be used to make up this medium. Autoclave and cool to 50 °C and then add the antibiotic to the desired concentration, mix and pour plates.

  • Lithium acetate (1.0 M) Dissolve 10.2 g of lithium acetate dihydrate in 100 ml of water bottle, autoclave for 15 min and store at room temperature (20 °C). This can also be filter sterilized using a Nalgene Filter Unit and a vacuum pump.

  • PEG MW 3350 (50% w/v) Add 50 g of PEG 3350 (Sigma Chemical Co. Ltd, cat. no. P-3640) to about 30 ml of distilled/deionized water in a 150 ml beaker. Stir until dissolved. Use a hot plate to gently warm the solution if necessary. Make the volume up to 100 ml in a 100 ml measuring cylinder and mix thoroughly. Transfer the solution to a glass storage bottle and autoclave for 15 min. Alternatively, the solution can be filter sterilized. The PEG can be stored at room temperature. The bottle must be securely capped to prevent evaporation, which will increase the concentration of PEG in the transformation reaction and severely reduce the yield of transformants. Make new liquid PEG every few months to avoid this problem. This can also be filter sterilized using a Nalgene Filter Unit and a vacuum pump; however, this may take a few hours.

  • Single-stranded carrier DNA (2.0 mg ml−1) Dissolve 200 mg of salmon sperm DNA (Sigma Chemical Co. Ltd, cat. no. D-1626) in 100 ml of sterile TE (10 mM Tris–HCl, 1 mM Na2 EDTA, pH 8.0) using a magnetic stir plate at 4 °C. This should take up to few hours. One can speed up the dissolution by drawing the DNA up and down a wide bore 25 ml pipette until no visible DNA is seen. There is no need to sonicate the carrier DNA as was described in earlier publications2. Larger molecular weight DNA was found to work more effectively as a carrier. Dispense 20 samples of 1.0 ml into 1.5 ml microcentrifuge tubes and the remainder in 5 ml samples in 15 ml screw-capped plastic centrifuge tubes and store at −20 °C. Denature the carrier DNA in a boiling water bath for 5 min and chill immediately in an ice/water bath before use. Denatured carrier DNA can be boiled three or four times without significant loss of activity.

  • Frozen competent cell solution Make a solution of 20% (v/v) sterile glycerol and sterilize in an autoclave. Dilute with sterile water to a final concentration of 5% (v/v) and 10% (v/v) sterile DMSO.

Procedure

Cell growth

  1. 1

    Yeast strains not containing any plasmids should be grown in rich medium (option A). Yeast strains containing a plasmid that requires selection should be grown on selective medium (option B).

    1. A

      Yeast cells without a plasmid

      1. i

        Inoculate your yeast strain into 25 ml of liquid 2 × YPAD medium and incubate overnight on a rotary shaker at 200 r.p.m. and 30 °C. Place a bottle of 2 × YPAD and two sterile 2 liter or a sterile 4 liter culture flask with baffles in the incubator as well.

    2. B

      Yeast strain with a plasmid

      1. i

        Inoculate your yeast strain containing a plasmid into 200 ml of the appropriate SC selection medium in a 2 liter sterile flask and incubate overnight on a rotary shaker at 200 r.p.m. and 30 °C.

  2. 2

    After 12–16 h of growth, determine the titer of the yeast culture. This can be done using a spectrophotometer (option A) or a hemacytometer (option B).

    1. A

      Using a spectrophotometer

      1. i

        ipette 10 μl of cells into 1.0 ml of water in a spectrophotometer cuvette, mix thoroughly by inversion and measure the OD at 600 nm (a suspension containing 1 × 106 cells ml−1 will give an OD600 of 0.1). Remember to multiply by the dilution factor to determine the titer in the cell culture.

    2. B

      Using a hemacytometer

      1. i

        Pipette 100 μl of suspension into 900 μl of sterile water in a microcentrifuge tube and mix thoroughly. Deliver 10 μl of this dilution onto the counting grid of an improved Neubauer hemacytometer, put the coverslip in place, wait several minutes for the cells to settle and count the number of cells in the 25 large grid squares using a microscope with a × 10 ocular and a × 10 objective lens. Multiply this number by 10,000 to obtain the titer in the diluted suspension. Remember to multiply by the dilution factor to determine the titer in the cell culture.

  3. 3

    Add 2.5 × 109 cells to 500 ml of the pre-warmed 2 × YPAD in the pre-warmed culture flask. The titer of this solution should be 5 × 106 cells ml−1.

  4. 4

    Incubate the flask in the shaking incubator at 30 °C and 200 r.p.m. until the cell titer is at least 2 × 107 cells ml−1. This should take about 4 h.

  5. 5

    Harvest the cells by centrifugation at 3,000g for 5 min, wash the cells in 0.5 volumes of sterile water, re-suspend in 0.01 volumes of sterile water, transfer to a suitable sterile centrifuge tube and pellet the cells at 3,000g for 5 min at 20 °C.

  6. 6

    Re-suspend the cell pellet in 0.01 volumes of filter sterile frozen competent cell (FCC) solution (5% v/v glycerol, 10% v/v DMSO). Use good quality sterile DMSO.

  7. 7

    Dispense 50 μl samples into an appropriate number of 1.5 ml microcentrifuge tubes.

  8. 8

    Place microcentrifuge tubes into a 100 tube styrofoam rack with lid. It is best to place this container upright in a larger box (Styrofoam or cardboard) with additional insulation such as Styrofoam chips or newspaper to reduce the air space around the sample box. This will result in the samples freezing slowly, which is essential for good survival rates.

  9. 9

    Put the large Styrofoam container in a −80 °C freezer overnight. The Styrofoam rack containing the frozen yeast cells can then be removed from the freezing container and stored at −80 °C.

    Pause point

    These cells can be stored for up to 1 year at −80 °C with little loss of transformation efficiency. Cells can be transformed using the protocol below.

Cell transformation

  1. 10

    Thaw cell samples in a 37 °C water bath for 15–30 s.

  2. 11

    Centrifuge at 13,000g in a microcentrifuge for 2 min and remove the supernatant.

  3. 12

    Make up frozen competent cell (FCC) transformation mix for the planned number of transformations plus one extra, according to the protocol described below. Include an extra tube for a negative control tube for no plasmid DNA. Add this to the pellet and vortex mix vigorously to re-suspend the cell pellet. Note the difference in PEG volume from all other procedures.

    Table 1 Table 2
    Full size table
  4. 13

    Incubate in a 42 °C water bath for 20–60 min depending on the strain. Temperature-sensitive strains can be left on the bench overnight and then carried on to the next step.

  5. 14

    Centrifuge the tubes at 13,000g for 30 s in a microcentrifuge and remove the supernatant with a micropipettor. Transformations utilizing plasmids with prototrophic gene selection use option A and for those utilizing plasmids with eukaryotic antibiotic genes use option B.

    1. A

      Prototrophic gene selection

      1. i

        Pipette 1.0 ml of sterile water into the transformation tube. Stir the pellet with a sterile micropipette tip to break up the cell pellet and then vortex mix to thoroughly re-suspend pellet.

    2. B

      Eukaryotic antibiotic gene selection

      1. i

        Pipette 1.0 ml of YPAD liquid medium into the transformation tube. Vortex mix to thoroughly re-suspend pellet.

      2. ii

        Incubate for 2–3 h at 30 °C to ensure good expression from the input plasmid DNA.

  6. 15

    Plate 2, 20 or 200 μl of the cell suspension onto the appropriate SC selection medium. The 2 and 20 μl volumes should be delivered into a puddle of 100–200 μl of sterile water or YPAD depending on the selection (see Step 14). Once delivered, the inoculum is then spread with a glass rod, made sterile by being soaked in ethanol and passed through the flame of a Bunsen burner or alcohol lamp. The volume plated will depend on the efficiency of your yeast strain. Allow the liquid to be absorbed into the medium by incubation at room temperature. Cells should be plated less densely when possible, as plating density negatively affects transformation efficiency.

  7. 16

    Incubate the plates at 30 °C for 3–4 days and recover the transformants.

    Troubleshooting

Troubleshooting

Troubleshooting advice can be found in Table 1.

Table 1 Troubleshooting table.
Full size table

Timing

Step 1: 12–16 h

Steps 2 and 3: 30 min

Step 4: approximately 4 h depending on the yeast strain and growth conditions

Steps 5–8: 1 h

Step 9: 12–16 h

Steps 10–14: 30 min to 1.25 h depending on the heat shock used.

Anticipated results

This protocol will yield up to 1 × 106 transformants per microgram of autonomously replicating plasmid DNA with a good transforming strain. Plasmids or DNA fragments requiring integration will be less efficient. The frozen competent yeast cells are stable for 6 months to a year with little loss in efficiency.