Agatep, Ronald

Gietz, R. Daniel

Kirkpatrick, Robert D.

Parchaliuk, Debra L.

Woods, Robin A.

Elsevier - Publisher Connector 

Technical Tips Online, Vol. 3, 1998
Transformation of Saccharomyces
cerevisiae by the lithium
acetate/single-stranded carrier
DNA/polyethylene glycol protocol
Ronald Agatep, Robert D. Kirkpatrick, Debra L. Parchaliuk, Robin A.
Woods* and R. Daniel Gietz
Department of Human Genetics, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E 0W3
Keywords: Cloning & sequencing
Introduction
▼Transformation of bacteria was first suggested by Grif-
fiths in 1928. It was not until fifty years later that a system
was reported by Hinnen et al. (Ref. 1) and Beggs (Ref. 2)
for the induction of transformation in Saccharomyces cere-
visiae. Further development by Ito et al. (Ref. 3) allowed
transformation of intact yeast cells following exposure to
alkali cations. This procedure was less complicated, but it
yielded only 400 transformants/µg of plasmid DNA. Schi-
estl and Gietz (Ref. 4) increased the efficiency of the al-
kali cation protocol to 100 000 transformants/µg plasmid
DNA by using single-stranded carrier DNA in the transfor-
mation mixture. Since this time, we have streamlined and
optimized this protocol to give yields as high as 2.2 × 107
transformants/µg DNA (Ref. 5, 6, 7, 8). High efficiency is es-
sential for transformation of cDNA expression libraries for
the two-hybrid system (Ref. 9, 10) as well as other similar
systems (Ref. 11, 12, 13).
Three transformation protocols are listed here. The ‘stan-
dard high-efficiency’ version of the lithium acetate (LiAc)/
single-stranded DNA (ss-DNA)/ polyethylene glycol (PEG)
protocol is used when a large number of transformants are
required. The ‘large-scale high efficiency’ version is used
to obtain the millions of transformants needed to screen
complex cDNA libraries. The ‘quick and easy’ version can be
used when large numbers of transformants are not required.
*Robin A. Woods is in the Department of Biology, University of Winnipeg,
515 Portage Ave, Winnipeg, Manitoba, Canada R3B 2E9.
Corresponding author: gietz@cc.umanitoba.ca
Solutions
Lithium acetate (1.0 M)
Dissolve 5.1 g of lithium acetate dihydrate (Sigma Chem-
ical Co. L-6883) in 50 ml sterile distilled deionized water
(ddH2O). Autoclave the solution and store it at room tem-
perature. To make 100mM stock LiAc, dilute 1 ml of 1.0 M
into 9 mls of sterile ddH2O.
Polyethylene glycol (PEG) MW 3350 (50% w/v)
Dissolve 50 g of PEG MW 3350 (Sigma Chemical Co. P-
3640) in 30 ml sterile ddH2O on a stirring plate. Adjust
the volume to 100 ml and autoclave. Store it in a se-
curely capped bottle at room temperature. Evaporation
from poorly sealed containers will increase the concentra-
tion of PEG and dramatically reduce transformation effi-
ciencies.
Single-stranded carrier DNA (2 mg/ml)
Dissolve 200mg salmon spermDNA (Sigma catalogue num-
ber L-1626) in 100 ml sterile TE buffer (10 mM Tris-HCl,
1 mM Na2 EDTA, pH 8.0) by stirring overnight at 4◦C. Dis-
pense 0.5 ml samples of the solution into 1.5 ml microfuge
tubes. Incubate the tubes in a boiling water bath for 5 min
and then chill in ice water. Store the samples at − 20◦C. Al-
ternatively, the double-stranded carrier DNA can be stored
at − 20◦C and when needed, boiled for 5 min and chilled in
ice water before use. Carrier DNA can be boiled, frozen and
thawed three or four times with negligible effect on trans-
formation efficiency, after which it should be discarded.
1366-2120 c©1998 Elsevier Ltd. All rights reserved. PII: S1366-2120(08)70121-1 www.sciencedirect.com 133
Technical Tips Online, Vol. 3, 1998 Technical Tips
Media
To condition the cells for transformation the yeast cells
are grown in YPD + adenine (YPAD) medium. Transformed
yeast cells are plated onto synthetic complete (SC) dropout
media to select the transformed plasmid. Recipes for both
are described in Gietz et al. (Ref. 10).
Standard high-efficiency transformation protocol (1 × scale)
The following procedure yields standard efficiencies of
greater than 1 × 106 transformants/µg plasmid DNA when
108 cells are transformed with 100 ng plasmid DNA. In-
creasing the amount of plasmid DNA reduces the trans-
formation efficiency but increases the transformation yield
or total number of transformants produced. Plasmid DNA
concentrations of up to 10 µg per transformation reaction
can be used, but it is usually more productive to ‘scale up’
the transformation reactions for these quantities of DNA
(Ref. 10).
Protocol 1: standard high-efficiency transformation
(1 × scale)
Day 1
1. Inoculate yeast strain into 5 ml of YPAD or 10 ml of the
appropriate SC-dropout medium and incubate overnight at
30◦C on a shaker at 200 rpm.
Day 2
2. Determine the titre of the overnight culture. This can
be done by measuring the OD600 of a 1 in 10 dilution into
water. For most strains an OD600 of 0.1 corresponds to ap-
proximately 1 × 106 cells/ml. Alternatively, the titre can be
determined using a haemocytometer.
3. Calculate the volume of overnight culture that con-
tains 2.5 × 108 cells and add to prewarmed (30◦C) YPAD
medium, to give a total volume of 50 ml in a 250 ml flask.
This will give a starting cell titre of about 5 × 106 cells / ml.
4. Incubate the diluted culture at 30◦C on a shaker at
200 rpm until the cells have gone through two divisions (2
× 107 cells/ml, which is sufficient for 10 transformations).
This may take 3−4 h, depending on the yeast strain.
5. Harvest the cells in a sterile 50ml disposable centrifuge
tube by centrifugation at 3000 × g for 5 min and remove
the supernatant.
6. Resuspend the cells in 25 ml of sterile ddH2O to wash
the cells, pellet again by centrifugation, and remove the
supernatant.
7. Resuspend the cell pellet in a approximately 900 µl of
sterile ddH2O and transfer to a 1.5 ml microcentrifuge tube.
8. Pellet the cells by centrifugation at 13 000 rpm for
1 min and resuspend the pellet in 100 mM LiAc to a final
volume of 1 ml. (Add approximately 700 µl 100 mM LiAc.)
Incubate at 30◦C for 10 min.
9. For each 1 × transformation reaction aliquot 100 µl of
the LiAc cell suspension into a new 1.5 ml microcentrifuge
tube. Pellet the cells at top speed in a microcentrifuge for
1 min. Remove the supernatant.
10. Add the following reagents in the order listed tomake
the transformation mix for each transformation planned:
50% PEG, 240 µl
1.0 M LiAc, 36 µl
ss-DNA (2 mg/ml), 50 µl
plasmid DNA, X µl*
sterile ddH2O, 34 µl − X µl*
total volume, 360 µl
*Note: the total volume of the 4th and 5th components
is 34 µl.
Vortex to mix and add the transformation mix to the
cell pellet and vortex vigorously until the pellet is fully
resuspended.
11. Incubate the transformation mixture at 30◦C for
30 minutes.
12. Heat shock the cells in a 42◦C water bath for 30
minutes.
13. Pellet the cells at top speed in a microcentrifuge for
1 min and remove the transformation mixture.
14. Add 1.0 ml of sterile ddH2O and resuspend by stir-
ring the cell pellet with micropipette tip or gently pipet-
ting the water up and down. Vortex briefly to ensure even
resuspension and plate each sample onto the appropriate
SC-dropout medium.
15. Incubate the plates at 30◦C for 2−4 days until colonies
appear.
Large-scale, high-efficiency transformation protocol
One and two-hybrid screens require a high yield of trans-
formants for good coverage of complex cDNA libraries. For
screens we use either a 30 × or a 60 × scale-up to ob-
tain an adequate number of transformants. Before a large-
scale transformation it is necessary to perform an experi-
ment at the 1 × scale with increasing amounts of library
DNA to determine the amount of plasmid DNA and the
specific scale of transformation needed for good library
coverage. Table 1 shows that it is more effective to do a
10 × scaled up with 10 × 1 µg of plasmid DNA than a 1
× transformation with 10 µg of plasmid DNA. In addition,
this data shows that a 30 × scale transformation with 30
µg of plasmid DNA will generate approximately 3.0 × 107
transformants, which is good coverage for a library with a
complexity of 1−2 × 106 independent clones.
134 www.sciencedirect.com
Technical Tips Technical Tips Online, Vol. 3, 1998
Table 1. Transformation efficiency vs
transformation yield
Plasmid TRAFO efficiency TRAFO yield
DNA transformants/mg transformants/reaction
0.1 µg 9.5 × 105 9.5 × 104
1.0 µg 1.1 × 106 1.1 × 106
10 µg 2.7 × 105 2.7 × 106
Yeast strain PJ69-4a was transformed with increasing amounts of
YEplac181 plasmid DNA (Ref. 15).
In most one- and two-hybrid screens two different plas-
mids, such as the GAL4 binding (GAL4BD) and GAL4 ac-
tivating domain (GAL4AD) fusion plasmids, are ultimately
transformed into your yeast strain. The best strategy to
optimize transformation yield is to initially transform the
yeast strain with the bait or target plasmid using the ‘quick
and easy’ protocol (see below). The plasmid library is then
transformed into the yeast strain containing the bait or tar-
get plasmid. This is done by growing an inoculum of the
plasmid-containing yeast selecting for maintainance of the
bait or target plasmid. This innoculum is then used to pro-
duce yeast cells that are competent for transformation by
growth for at least two generations in a rich non-selective
medium such as YPAD. This short growth in non-selective
medium does not usually result in significant plasmid loss.
These cells are then transformed with library plasmid DNA
(up to 200 µg) to produce enough transformants for most
screens. The following protocol is for a 30 × or 60 × scale-
up of the ‘high efficiency’ protocol, however a 120 × scale
can be performed if desired.
Protocol 2: one or two-hybrid screening
Day 1
1. Inoculate the yeast strain containing the first plasmid
into the appropriate SC-drop-out medium (usually SC-Trp)
in an appropriate sized flask and incubate at 30◦C overnight
(16−24 h) on a shaker at 200 rpm. For a 30 × scale-up, use
50 ml medium in a 500 ml flask; for a 60 × scale-up, use
100 ml medium in a 1 l flask.
Day 2
2. Determine the titre of the overnight culture. This can
be done by measuring the OD600 of a 1 in 10 dilution. For
most strains an OD600 of 0.1 corresponds to approximately
1 × 106 cells/ml. Alternatively, the titre can be determined
using a haemocytometer.
3. Calculate the volume of overnight culture that con-
tains the number of cells needed (Table 2). Harvest that
volume of SC-dropout culture by centrifugation and resus-
Table 2. Culture volumes (SC-dropout media) for
TRAFO culture
30 × scale 60 × scale
Number of cells neededa 7.5 × 108 cells 1.5 × 109cells
Volume of SC-Trpb 37.5 ml 75 ml
Volume of YPAD 150 ml 300 ml
Volume of flask 1 l 2 l
aVolume of TRAFO culture (150 or 300 ml) × 5 × 106 cells/ml.
bVolume of SC-Trp culture = number cells needed / cell titer
(cells/ml) of SC-Trp culture.
Table 3. Lithium acetate volumes
30 × scale 60 × scale
100 mM LiAc 3 ml 6 ml
Centrifuge tube 15 ml 15 ml
pend the cells in the appropriate volume of YPAD medium
which has been pre-warmed to 30◦C. This will give a start-
ing cell titre of 5 × 106 cells/ml. In most cases the SC-Trp
culturewill give a cell titer of approximately 2× 107 cells/ml
in an overnight culture.
4. Incubate the YPAD culture at 30◦C and 200 rpm until
the cells have gone through 2 divisions, which may take
3−4 h, depending on the strain. The cell titre should be
at least 2 × 107 cells/ml which corresponds to an OD600 of
approximately 2.0.
Note: always measure the OD600 of this culture after a
1/10 dilution.
5. Harvest the cells in 3 or 6 sterile 50 ml disposable
centrifuge tubes by centrifugation at 3000 × g for 5 min
and remove the supernatant.
6. Resuspend the cells from each 50 ml centrifuge tube
with 25 ml of sterile ddH2O and pellet again by centrifuga-
tion and remove the supernatant.
7. Resuspend and pool thewashed cells in the appropriate
volume of 100 mM LiAc (Table 3) and incubate in a water
bath at 30◦C for 15 min.
8. Transfer the cells in a 50 ml disposable centrifuge tube,
harvest by centrifugation at 3000 × g for 5 min, and discard
the supernatant.
9. Add, in the order indicated, the transformation mix
components listed in (Table 4) to a separate tube and mix
thoroughly by vortexing. Add the mixed transformation
mix to the cell pellet and vortex vigorously to resuspend
the cell pellet in the transformation mix.
10. Incubate in a water bath at 30◦C for 30 min. Mix the
tube gently several times after 10 and 20 min to mix the
contents thoroughly.
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Technical Tips Online, Vol. 3, 1998 Technical Tips
Table 4. Transformation mix components
Order 30 × scale 60 × scale
1st * 50% PEG 7.20 ml 14.40 ml
2nd* 1.0 M LiAc 1.08 ml 2.16 ml
3rd*ss-DNA (2 mg/ml) 1.50 ml 3.00 ml
4th* Plasmid DNA A ml B ml
5th* sterile ddH2O 1.02 − A ml* 2.04 − B ml*
Note: the amount of sterile ddH2O, plasmid DNA can
each vary but the total amount must not exceed the
volume listed in the 5th row.
Table 5. Volume of sterile ddH2O for
resuspension and plating
30 × scale 60 × scale
Volume of H2O 20 ml 40 ml
Plating volume 400 µl 400 µl
Number of150 mm plates 50 100
11. Heat shock the cells in a 42◦C water bath for 45 min.
Invert the tube gently several times at 5 min intervals to
mix the contents and aid in temperature equilibration.
12. Centrifuge at 3000 × g for 5 minutes and remove the
transformation mix.
13. Resuspend the cells in the volume of sterile water
listed in (Table 5) and plate 400 µl samples onto each plate
of selective medium. For two hybrid screens we resuspend
the cells in a total volume of either 20 or 40 mls and plate
400µl onto each of either 50 (30 ×) or 100 (60 ×) 150 mm
plates of SC-Trp, Leu, His (containing 3AT) medium, repec-
tively.
14. Incubate at 30◦C for 4−7 days at 30◦C.
We have successfully scaled up this protocol to 120 ×
scale. With the large volumes of cells and reagents it is
important to mix the tubes during all incubations at 30◦C
and 42◦C to ensure good temperature transfer.
Quick and easy transformation
This protocol is used when only a few transformants for a
specific plasmid are desired, such as for a GAL4BD bait plas-
mid or a one-hybrid target plasmid. Yeast grown overnight
on agar medium can be transformed up to an efficiency of
1 × 104 transformants / µg plasmid DNA. This is usually
more than adequate for the isolation of a transformant for
subsequent work.
1. Grow the culture to be transformed overnight on
YPAD, YPD or appropriate SC-minus medium.
2. Scrape approximately a 25 µl volume of cells from
the plate with a sterile tooth pick or inoculating loop and
resuspend in 1.0 ml of sterile water in a 1.5 ml microfuge
tube.
Note: To determine the size of a 25 µl volume of yeast,
scrape a blob of yeast using a sterile tooth pick or loop into
100 µl of sterile water and resuspend by vortexing. Measure
the resulting volume with a micropipette to determine the
volume contributed by the yeast cells.
3. Vortex vigorously to wash the cells.
4. Pellet the cells at top speed in a microcentrifuge for 30
sec.
5. Add to the cell pellet in the following order:
240 µl PEG
36 µl 1.0 M LiAc
50 µl ss-DNA
34 µl containing plasmid DNA (0.1−5.0 µg) and sterile
ddH2O, giving a final volume of 360 µl.
Vortex vigorously until resuspended.
6. Heat shock the cells in a 42◦C water bath for 30 min.
7. Pellet the cells at top speed in a microcentrifuge for 1
min and remove the transformation mixture.
8. Add 400 µl of sterile ddH20 to the pellet and incubate
at room temperature for 5 min. Resuspend by votexing or
gently pipetting the fluid up and downwith amicropipette.
Plate 200 µl samples onto 2 plates of the appropriate SC-
dropout medium.
9. Incubate at 30◦C for 2−4 days.
This protocol can be applied to cultures which have been
stored for several days or weeks. However, the yield of trans-
formants is best with fresh cultures.
Common problems and their solutions
Transformation efficiency and yield can be affected by a
number of factors. Perhaps the most important is the yeast
strain. Some strains transform well while others transform
with a much reduced efficiency (Ref. 6) Efficiency can be
improved by optimizing the amount of plasmid and ss-
carrier DNA and by increasing or decreasing the duration
of the heat shock at 42◦C (Ref. 7). If these fail to produce
an increase in transformation efficiency or yield, it is best
to consider using another yeast strain.
The growth of a culture to logarithmic phase is also im-
portant. Two cell divisions appear to be required for op-
timal transformation competence in yeast, however effi-
ciency remains high for an additional two divisions. Yeast
cells should be grown with vigorous aeration. Baffle flasks
(BellcoTM) produce better aeration than standard conical
flasks but are not essential.
The transformation efficiency and yield can also be af-
fected by the preparation and storage of the SC-drop-out
136 www.sciencedirect.com
Technical Tips Technical Tips Online, Vol. 3, 1998
medium. The pH of the medium should be adjusted to 5.6
before the addition of the agar and it should be autoclaved
for no more than 20 min. Some components of SC medium
are light sensitive and liquid and plates should be stored in
the dark at all times.
PlasmidDNA can be purified by numerousmethodswith-
out affecting its transformation characteristics. The same
amount of plasmid DNA purified by the miniprep method
transforms just as effectively as that purified by the CsCl
density gradient method. The size of the plasmid also af-
fects transformation, smaller plasmids are more effective
than large ones. We have not tested our protocols for YAC
transformation.
References
1 Hinnen, A., Hicks, J.B. and Fink, G.R. (1978) Proc. Natl. Acad. Sci. U.S.A.
75, 1929–1933.
2 Beggs, J.D. (1978) Nature 275, 104–109.
3 Ito, H., Fukuda, Y., Murata, K. and Kimura, A. (1983) J. Bacteriol. 153,
163–168.
4 Schiestl, R.H. and Gietz, R.D. (1989) Curr. Genet. 16, 339–346.
5 Gietz, D., Jean, A., Woods, R.A. and Schiestl, R.H. (1992) Nucleic Acids
Res. 20, 1425.
6 Schiestl, R.H., Manivasakam, P., Woods, R.A. and Gietz, R.D. (1993)
Methods: A Companion to Methods in Enzymol. 217, 478–483.
7 Gietz, R.D. andWoods, R.A. (1994) Johnston, J.R. (Ed.).Molecular Genet-
ics of Yeast: A Practical Approach, Oxford University Press, pp. 121–134.
8 Gietz, R.D., Schiestl, R.H., Willems, A. and Woods, R.A. (1995) Yeast 11,
355–360.
9 Fields, S. and Song, O. (1989) Nature 340, 245–246.
10 Gietz, R.D. et al. (1997) Mol. Cell. Biochem. 172, 67–79.
11 Wang, M.M. and Reed, R.R. (1993) Nature 364, 121–126.
12 Li, J.J. and Herskowitz, I. (1993) Science 262, 1870–1874.
13 Lui, J., Wilson, T.E., Milbrant, J. and Johnston, M. (1993) Methods: A
Companion to Methods in Enzymol. 5, 125–137.
14 James, P., Halladay, J. and Craig, E.A. (1996) Genetics 144, 1425–1436.
15 Gietz, R.D. and Sugino, A. (1988) Gene 74, 527–534.
Products Used
lithium acetate dihydrate: lithium acetate dihy-
drate from Sigma
PEG MW 3350: PEG MW 3350 from Sigma
sperm DNA: sperm DNA from Sigma
Baffle flasks: Baffle flasks from Bellco Glass Inc
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Transformation of Saccharomyces cerevisiae by the lithium acetate/single-stranded carrier DNA/polyethylene glycol protocol 

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Transformation of Saccharomyces cerevisiae by the lithium acetate/single-stranded carrier DNA/polyethylene glycol protocol

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