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High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method R Daniel Gietz1 & Robert H Schiestl2 1Department

of Biochemistry and Medical Genetics, University of Manitoba, T250-770 Bannatyne Ave., Winnipeg, Manitoba R3E 0W3, Canada. 2Department of Pathology, Environmental Health and Radiation Oncology, UCLA School of Public Health and David Geffen School of Medicine, 650 Charles E. Young Drive South, Los Angeles, California 90095, USA. Correspondence should be addressed to R.H.S. ([email protected]).

© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

Published online 31 January 2007; doi:10.1038/nprot.2007.13

Here we describe a high-efficiency version of the lithium acetate/single-stranded carrier DNA/PEG method of transformation of Saccharomyces cerevisiae. This method currently gives the highest efficiency and yield of transformants, although a faster protocol is available for small number of transformations. The procedure takes up to 1.5 h, depending on the length of heat shock, once the yeast culture has been grown. This method is useful for most transformation requirements.

INTRODUCTION The transformation of Saccharomyces cerevisiae with alkali cations was first described in 1983 by Ito et al.1 Many improvements have taken place over the past 20 odd years, making the procedure more efficient in generating transformants as well as shortening the procedure. The most significant improvement was the inclusion of single-stranded DNA as carrier, giving rise to an increase in transformation efficiency2. This technique has been optimized for cell number, carrier DNA concentration and plasmid DNA concentration3. This transformation method can be modified and used for different purposes for yeast molecular biology4. The highefficiency transformation protocol can be employed to screen

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 Na2EDTA, 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) EQUIPMENT . 1.5 ml polypropylene microcentrifuge tubes (Fisher Scientific Ltd, cat. no. 05-669-32). Tubes 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 . 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 15 mm sterile disposable Petri plates (Fisher Scientific Ltd, cat. no. 08-757-9B) . Nalgene Filter Unit 0.20 mm 500 ml (Fisher Scientific Ltd, cat. no. 50-0020/ EMD) . Microscope with an at least 10 objective and a 10 ocular lens. This is used with the hemacytometer to titer yeast cultures

various types of plasmid libraries. In addition, this protocol can be used to transform linear DNA constructs for DNA knockout experiments as well as oligonucleotides in yeast5–6. The various S. cerevisiae transformation methods were recently reviewed7 and the reader should refer to that document for a history of yeast transformation methods. This protocol can also be used for transformation of other yeast species with some modification7. If transforming a small number of transformants, a quicker and easier protocol is available8. Alternatively, the protocol can be scaled up using the library screen transformation protocol9 or a 96-well plate10.

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

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

. Precision Low Temperature Incubator Model 815. This is set at 30 1C 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). This is for growing liquid cultures. This is placed in the 30 1C incubator. This is a less expensive alternative to a floor model shaking incubator . Precision Water Bath Model 181 (Fisher Scientific Ltd, cat. no. 15-474-10) is used for 42 1C heat shock . A magnetic stir plate . 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 S. 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 high efficiency of transformations. 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 NATURE PROTOCOLS | VOL.2 NO.1 | 2007 | 31


PROTOCOL 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 grew the yeast in YPAD medium (1% (w/v) Bacto yeast extract, 2% (w/v) Bacto peptone, adenine hemisulfate 80 mg l 1) (for plates add 18 g l 1 Bacto Agar). The yeast cells to be transformed are usually regrown 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) or mixed according to the protocol of Rose11. 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 enable cells to grow in the presence of the eukaryotic antibiotic G418 or geneticin. This antibiotic is added to YPD plates at a concentration of 200–300 mg ml 1 depending on the strain being used. The ingredients for YPAD listed above, omitting adenine hemisulfate, can be used to make up this medium. Autoclave and cool to 50 1C and then add the antibiotic to the desired concentration, mix and pour plate. Lithium acetate (1.0 M) Dissolve 102 g of lithium acetate dihydrate in 100 ml of water in a bottle, autoclave for 15 min and store at room temperature (20 1C). 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 to about 30 ml of distilled/ deionized water in a 150 ml beaker. Stir until it dissolves. Use a hot plate to gently warm the solution if necessary. Make up the volume 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. Prepare

fresh 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 in 100 ml of sterile TE (10 mM Tris–HCl, 1 mM Na2EDTA, pH 8.0) using a magnetic stir plate at 4 1C. This should take up to a 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. Higher 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 1C. 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. Equipment setup Glassware . 250 ml baffled Erlenmyer flask, sterile (Fisher Scientific Ltd, cat. no. 10-0415B). This flask is used to grow the 50 ml YPAD yeast culture for transformation for good aeration. Cap with aluminum foil and autoclave. . Hemacytometer (Fisher Scientific Ltd, cat. no. S17040). This is used to titer the cultures. . Spectrophotometer (Fisher Scientific Ltd, cat. no. S42475P). This is used at 600 nm for OD for an alternate method of determining yeast cell numbers in cultures and used with plastic cuvettes (Fisher Scientific Ltd, cat. no. 14-385-938). . Glass rod bent to a P shape. The rod is made from a glass rod 6 inches in length and bent to a P shape so that the right-hand part of the P is about 1 inch in length. This is used to spread yeast cells onto plate in sterile condition.

PROCEDURE 1| Inoculate a single colony of the yeast strain with a sterile inoculation loop from a fresh YPAD plate into 5 ml of liquid medium (YPAD or appropriate SC selection medium) and incubate overnight on a rotary shaker at 200 r.p.m. and 30 1C. Place a bottle of 2 YPAD and a 250 ml culture flask in the incubator as well. 2| After 12–16 h of growth, determine the titer of the yeast culture. This can be performed by using a spectrophotometer (A) or a hemacytometer (B). (A) Using a spectrophotometer (i) Pipette 10 ml 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. (B) Using a hemacytometer (i) Pipette 100 ml of suspension into 900 ml of sterile water in a microcentrifuge tube and mix thoroughly. (ii) Deliver 10 ml of this dilution onto the counting grid of a hemacytometer, place the coverslip, wait for sometime 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 in cells per ml. 3| Add 2.5 108 cells to 50 ml of pre-warmed 2 YPAD in a pre-warmed culture flask. The titer of this solution should be 5 106 cells ml 1. 4| Incubate the flask in the shaking incubator at 30 1C and 200 rpm until the cell titer is at least 2 107 cells ml 1. This should take about 4 h. 5| Denature a 1.0 ml sample of carrier DNA in a boiling water bath for 5 min and chill immediately in an ice/water bath. Alternatively, a pre-denatured carrier DNA sample stored at 20 1C can be used, thawed and kept on ice until needed. 6| Harvest the cells by centrifugation at 3,000g for 5 min and resuspend the pellet in 25 ml of sterile water and centrifuge at 3,000g for 5 min at 20 1C to pellet the cells. Repeat this wash with another 25 ml of sterile water by resuspending the cells and pelleting them again by centrifugation. Resuspend the cells in 1.0 ml of sterile water. 7| Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge for 30 s at 13,000g and discard the supernatant. 32 | VOL.2 NO.1 | 2007 | NATURE PROTOCOLS

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8| Resuspend the cells in 1.0 ml of sterile water and pipette 100 ml samples containing 108 cells into 1.5 ml microcentrifuge tubes, one for each transformation. Centrifuge in a microcentrifuge at 13,000g for 30 s and remove the supernatant. For a single plasmid in each transformation reaction, follow option A. If there are different plasmids for each transformation reaction, follow option B. (A) Single plasmid (i) For a single plasmid in each transformation reaction, make up sufficient transformation mix for the planned number of transformations, plus one, extra using the recipe below. Mix thoroughly on a vortex mixer. Transformation mix components PEG 3350 (50% (w/v)) LiAc 1.0 M Single-stranded carrier DNA (2.0 mg ml 1) Plasmid DNA plus sterile water Total volume

Volume (ll) 240 36 50 34 360

(ii) Add 360 ml of transformation mix to each transformation tube and resuspend the cells by vortexing vigorously. Always include a negative control tube containing no plasmid unless you have experience with the yeast strain you are using. (B) Multiple plasmids (i) For different plasmids for each transformation reaction, make up sufficient transformation mix for the planned number of transformations plus an extra mix using the recipe below. Transformation mix components PEG 3350 (50% (w/v)) LiAc 1.0 M Single-stranded carrier DNA (2.0 mg ml 1) Total volume

Volume (ll) 240 36 50 336

(ii) Add 336 ml of transformation mix to each transformation tube containing the cell pellet. Add the plasmid DNA to each of the tubes plus water to a final volume of 34 ml. Always include a negative control tube containing no plasmid DNA unless you have experience with the yeast strain you are using. (iii) Resuspend the cell pellet by vortex mixing vigorously. 9| Place the tubes in a water bath at 42 1C and incubate for 40 min. Each strain may have a different optimum heat-shock time. Consider testing each strain for its optimum heat-shock time. Temperature-sensitive strains can be left on the bench overnight and then carried on to the next step. 10| 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 follow option A and those utilizing plasmids with eukaryotic antibiotic genes follow option B. (A) Prototrophic gene selection (i) Pipette 1.0 ml of sterile water into the transformation tube. Stir the pellet with a sterile micropipette tip to break the cell pellet and then vortex mix to uniformly resuspend pellet. (B) Eukaryotic antibiotic gene selection (i) Pipette 1.0 ml of YPD liquid medium into the transformation tube. Vortex mix to resuspend pellet. (ii) Incubate for 2–3 h at 30 1C to ensure good expression from the input plasmid DNA. 11| Plate 2, 20 or 200 ml of the cell suspension onto the appropriate SC selection medium. The 2 and 20 ml volumes should be delivered into a puddle of 100–200 ml of sterile water or YPD depending on the selection (see Step 12). Once delivered, the inoculum is spread with a glass rod, sterililized by an ethanol soak 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. 12| Incubate the plates at 30 1C for 3–4 days and count the number of transformants. ? TROUBLESHOOTING

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PROTOCOL ? TROUBLESHOOTING Troubleshooting advice can be found in Table 1.


TABLE 1 | Troubleshooting table. Problem Poor transformation efficiency

Possible reason Poor growth due to poor aeration

Solution Use a baffled flask for growth or only use 10% culture volume in a non-baffled flask

Poor transformation efficiency

PEG reagent evaporation

Prepare fresh PEG reagent, as evaporation of water changes the concentration and causes poor transformation

Poor transformation efficiency Poor transformation efficiency

Non-optimal heat-shock time Cells not grown for at least two cell divisions

Poor transformation efficiency

Degraded carrier DNA

Test your strain for the optimal heat-shock time Utilize 2 YPAD medium and grow the cells for at least two divisions. Good growth is essential for transformation. Cells grown for three or four divisions will also transform efficiently Denature a fresh tube of carrier. Freeze/thaw only 3–4 times

No transformation

Cells plated onto incorrect medium

Check the requirements for your strain and ensure that your medium contains each of the required nutrients

Plate is confluent with growth

Contamination of culture with bacteria or other microbe

Streak out your stock yeast culture to ensure no contamination is present. Check liquid medium for contamination


Yeast cells were plated onto wrong medium

Check the genotype of your strain, plasmid marker and medium used


Wrong dilution was plated

Always plate a range of dilutions to ensure isolation of individual transformants

TIMING Step 1, 12–16 h; Steps 2 and 3, 30 min; Step 4, 4 h depending on the strain and growth conditions; Steps 5–11, 1–1.5 h depending on heat-shock duration; Step 12, 3–4 days. Yeast transformants require at least 3 days to produce sizeable colonies. ANTICIPATED RESULTS This protocol will generate up to 1 106 transformants per microgram of autonomously replicating plasmid DNA with a good transforming strain. Fewer transformants will be generated using integrating plasmids and DNA fragments requiring genome integration. A highly efficient yeast strain will give up to 200 transformants on a plate with 2 ml plated cells/medium. With most strains, you can anticipate up to 1 106 transformants per mg plasmid DNA per 108 cells.

COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Published online at http://www.natureprotocols.com Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions 1. Ito, H., Fukuda, Y., Murata, K. & Kimura, A. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163–168 (1983). 2. Schiestl, R.H. & Gietz, R.D. High efficiency transformation of intact yeast cells using single-stranded nucleic acids as carrier. Curr. Genet. 16, 339–346 (1989). 3. Gietz, R.D., Schiestl, R.H., Willems, A.R. & Woods, R.A. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11, 355–360 (1995). 4. Gietz, R.D. & Woods, R.A. Yeast transformation. in Methods in Enzymology, Guide to Yeast Genetics and Cell Biology, Parts B and C, Vol. 350 (eds. Guthrie, C. & Fink, G.R.) 87–96 (Academic Press, San Diego, CA, 2001).

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5. Linske-O’Connell, L.I., Sherman, F. & McLendon, G. Stabilizing amino acid replacements at position 52 in yeast iso-1-cytochrome c: in vivo and in vitro effects. Biochemistry 34, 7094–7102 (1995). 6. Yamamoto, T., Moerschell, L.R.P., Wakem, P., Ferguson, D. & Sherman, F. Parameters affecting the frequencies of transformation and co-transformation with synthetic oligonucleotides in yeast. Yeast 8, 935–948 (1992). 7. Gietz, R.D. & Woods, R.A. Genetic transformation of yeast. BioTechniques 30, 816–831 (2001). 8. Gietz, R.D. & Schiestl, R.H. Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protocols doi: 10.1038/nprot. 2007.14. 9. Gietz, R.D. & Schiestl, R.H. Large scale high efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protocols doi: 10.1038/ nprot.2007.15. 10. Gietz, R.D. & Schiestl, R.H. Microtiter plate transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protocols doi: 10.1038/nprot.2007.16. 11. Rose, M.D. Isolation of genes by complementation in yeast. Methods Enzymol. 152, 481–504 (1987).