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. 14: 565–571 (1998)
An Improved Protocol for the Preparation of Yeast
Cells for Transformation by Electroporation
Department of Biochemistry, Merck & Co., Rahway, NJ 07065, U.S.A.
Received 10 July 1997; accepted 9 November 1997
Pretreatment of yeast cells with lithium acetate (LiAc) and dithiothreitol (DTT) enhances the frequency of
transformation by electroporation. The method shows improvements of 6–67-fold in wild-type strains derived from
commonly used Saccharomyces cerevisiae genetic backgrounds. In addition, 15–300-fold improvement in transformation frequency was achieved with several mutant strains of S. cerevisiae that transformed poorly by conventional
procedures. Both DTT and lithium acetate were necessary for maximal transformation frequencies. Pretreatment
with lithium and DTT also resulted in an 3·5-fold increase in the electroporation transformation frequency of the
pathogenic fungus Candida albicans. 1998 John Wiley & Sons, Ltd.
Yeast 14: 565–571, 1998.
  — Saccharomyces cerevisiae; electroporation; transformation
A standard approach to characterizing novel
mutants of Saccharomyces cerevisiae involves
identification of the target gene by complementation cloning. However, a common technical
hurdle is often encountered when the mutant
under study exhibits a secondary phenotype of
poor transformation efficiency. This makes it difficult to obtain the large number of transformants
necessary to thoroughly cover a typical genomic
library. Achieving high transformation frequencies
for the pathogenic fungus Candida albicans is also
challenging because an autonomously replicating
sequence that is as efficient as the S. cerevisiae 2ì
element (Kurtz et al., 1987; Cannon et al., 1990;
Herreros et al., 1992) is not available for Candida
species. As a consequence, integrative transformation is much more widely used for C. albicans than
*Correspondence to: R. Kelly, R80Y-200, Merck & Co., PO
Box 2000, Rahway, NJ 07065, USA. Tel: (+1) 908 594 6385;
fax: (+1) 908 594 5468; e-mail:
CCC 0749–503X/98/060565–07 $17.50
1998 John Wiley & Sons, Ltd.
for S. cerevisiae. C. albicans is usually transformed
by a method requiring time-consuming formation
of spheroplasts or by less efficient lithium acetate
protocols which often require large amounts of
DNA (Kurtz and Scherer, 1991). We tried to
improve transformation of C. albicans using
modifications that improve lithium acetate transformation of S. cerevisiae, i.e. the inclusion of
either DMSO (Hill et al., 1991) or single-stranded
carrier DNA (Schiestl and Gietz, 1989). However,
in our hands, neither of these methods enhanced
transformation of C. albicans.
While attempting to identify the target gene
producing resistance to a new antifungal compound, we encountered a S. cerevisiae mutant
that was almost completely refractory to transformation by published electroporation (Becker
and Guarente, 1991) or lithium acetate (Ito et al.,
1984; Chen et al., 1992) procedures. This paper
describes a simple modification to an established
electroporation protocol that provides approximately 1–2 orders of magnitude increase in
transformation efficiency of three mutant strains as
well as two wild-type S. cerevisiae strains that are
derived from commonly used genetic backgrounds.
The new method also improves the electroporation
transformation efficiency of a strain of C. albicans.
Strains and plasmids
YPH98 (MATa ura3–52 lys2–801 ade2–101
trp1-Ä1 leu2-Ä1) was provided by P. Hieter
(Sikorski and Hieter, 1989). W303–1A (MATa
ade2–1 can1–100 his3–11,15 leu2-3,112 trp1
ura3–1) was provided by R. Rothstein (Thomas
and Rothstein, 1989). Three poorly transforming
S. cerevisiae strains were employed in the
study. R812–5 is a spontaneous temperaturesensitive mutant of YPH98, selected for resistance
to a new antifungal compound. The R812–5 ts
and drug resistance phenotypes are both
complemented by a plasmid containing a wild-type
SIN4 gene (data not shown). GPY1103 (MATa
leu2–3,112 ura3–52 his4–519 trp1 can1 chc1–
Ä8::LEU2) is a strain disrupted for the clathrin
heavy chain (Payne et al., 1988). MY2201 (MATa
ade2–1 can1–100 his3–11,15 leu2–3,112 ura3–1
erg6::LEU2) is an erg6 disruption described by
Gaber et al. (1989) and transplaced in our lab into
the W303 genetic background. C. albicans CAI4
(Äura3::imm434/Äura3::imm434) was kindly provided by William Fonzi (Fonzi and Irwin, 1993).
YEplac195 is a 2ì-based replicating plasmid with
a URA3 nutritional marker (Gietz and Sugino,
1988). Plasmid pRS316 is a centromeric vector
with a URA3 nutritional marker (Sikorski and
Heiter, 1989). The C. albicans URA3 gene (Gillum
et al., 1984) was independently cloned by complementation of the ura3 mutation of S. cerevisiae
W303–1A (MATa, ura3–1 leu2–3,112 his3–11
trp1–1 ade2–1) with a genomic library constructed
in YEp13 (Rosenbluth et al., 1985). Plasmid
pJAM11 is a C. albicans URA3 subclone containing an 4·5 kb XbaI fragment in vector pUC18.
All media were standard for yeast genetics and
described in Sherman et al. (1986).
Nucleic acid isolations and hybridizations
Plasmid DNA was isolated using the QIAGENtip 500 or 100 procedure (QIAGEN Inc.,
Chatsworth, CA). Genomic DNA of C. albicans
1998 John Wiley & Sons, Ltd.
. .   .
was isolated by the glass bead lysis method of
Hoffman and Winston (1987). The Southern blot
was performed with a Zeta-ProbeGT derivatized
nylon membrane (Bio-Rad, Richmond, CA)
and was hybridized under stringent conditions
recommended by the manufacturer. The probe
was radiolabeled with [á-32P]dCTP using a
random-primed DNA labeling kit (Stratagene, La
Jolla, CA).
DNA-mediated transformation by electroporation
Both S. cerevisiae and C. albicans were grown
overnight to stationary phase. Fifty microliters of
the S. cerevisiae culture were inoculated into
100 ml of YPAD broth and 0·1 A600 units of the
C. albicans culture were inoculated into 100 ml
YPAD broth containing 0·4 m uridine and
grown overnight. S. cerevisiae was harvested at an
A600 nm of 0·7–1·5 and C. albicans was harvested
at an A600nm of 1·3. C. albicans cells from the
early logarithmic stage of growth were optimal for
transformation. The cell pellets were collected by
centrifugation, suspended in 25 ml of 0·1 -lithium
acetate, 10 m-dithiothreitol, 10 m-Tris–HCl,
pH 7·5, 1 m-EDTA (LiAc/DTT/TE) and incubated at room temperature for 1 h. Subsequent
steps were similar to the S. cerevisiae protocol of
Becker and Guarente (1991). The cells were
collected by centrifugation at 5000 rpm and the
pellet was suspended in 25 ml ice-cold water. This
step was repeated and the pellet was then suspended in 10 ml of ice-cold 1 -sorbitol. Following
centrifugation, the pellet was suspended in 100 ìl
of 1 -sorbitol which yields 500 ìl of cell suspension. Forty microliters of cell suspension were used
for each transformation. This corresponds to
4–8108 cells. Plasmid DNA was added to the
cell suspension and incubated on ice for 5 min. The
mixture was transferred to a 0·2 cm electroporation cuvette and pulsed at 1·5 kV, 25 ìFD, 200
ohms with a BioRad gene pulser. Immediately,
1 ml cold 1 -sorbitol was added and the contents
of the cuvette were gently mixed. One-hundred
microliter aliquots of the electroporated cell suspensions, or appropriate dilutions thereof, were
plated. Ura + transformants were selected on synthetic medium containing Yeast Nitrogen Base
without amino acids supplemented with 2·0% glucose and 0·87% -Ura dropout powder (BIO101).
S. cerevisiae colonies were counted after 3 days
incubation at 30C and C. albicans transformants
were selected after incubation for 4–5 days at 30C.
. 14: 565–571 (1998)
    
Table 1.
Enhanced transformation efficiency of poorly transforming Saccharomyces
1, 3 (2 ng)
294, 258 (2 ng)
3 (10 ng)
125 (2 ng)
86 (10 ng)
254 (2 ng)
Each transformation was performed with 100 ng of pRS316. The results of two independent
transformations are reported for strain R812–5. The number in parentheses indicates the amount of
DNA plated.
Table 2. Electroporation transformation efficiency of wild-type S. cerevisiae strains with
various pretreatments.
Strain YPH98
Strain W303–1A
per micrograma
per micrograma
Each transformation included 100 ng YEplac195 DNA. For quantitation of these transformants
serial dilutions of each transformation were plated and dilutions yielding 50–200 colonies were used to
calculate transformants/ìg; nt, not tested.
Enhanced transformation of a poorly transforming
S. cerevisiae mutant
Faced with the obstacle of optimizing transformation of a poorly transforming mutant, we
reasoned that treatments that normally induce
cells to take up DNA might also enhance the
electroporation efficiency. We therefore modified a
standard electroporation protocol to include pretreatment for 1 h with a solution of LiAc/DTT/TE.
Lithium ions are known to enhance yeast transformation (Ito et al., 1984) and more recently several
labs have shown improvements with the addition
of DTT (Meilhoc et al., 1990; Becker and
Guarente, 1992; Chen et al., 1992). However, to
our knowledge, a combination of both lithium
acetate and DTT has never been used in an
electroporation protocol. We found that the LiAc/
DTT/TE pretreatment resulted in an approximately 1–2 order of magnitude enhancement of the
1998 John Wiley & Sons, Ltd.
electroporation transformation efficiency in poorly
transforming S. cerevisiae mutants (Table 1).
Does LiAc/DTT treatment enhance transformation
in wild-type strains?
We evaluated whether the enhancement of
electroporation transformation efficiency by the
LiAc/DTT/TE treatment is limited to mutant
strains or represents a general means of improving
transformation in Saccharomyces strains. We
examined the behavior of two wild-type strains
derived from genetic backgrounds in common use
within the Saccharomyces research community.
Strain YPH98 is isogenic with S288C (Sikorski and
Hieter, 1989) and is the wild-type parent of mutant
R812–5. The other wild-type strain evaluated was
W303–1A. As shown in Table 2, pretreatment
with LiAc/DTT/TE improved the transformation
frequency more than treatment with either
component alone.
. 14: 565–571 (1998)
. .   .
Table 3. Electroporation transformation efficiency of C. albicans CAI4 with various
Transformants/ìg linear DNAa
Exp. 1
Exp. 2
Exp. 3
Average no.
Average foldenhancement
Transformants/ìg circular DNAb
Exp. 1
Exp. 2
Exp. 3
Average no.
Each transformation included 0·5 ìg HpaI digest of pJAM11 DNA. bEach transformation included
1·0 ìg pJAM11 DNA.
Enhanced transformation of a C. albicans ura3
We also tested the effect of LiAc/DTT pretreatment on the electroporation transformation
efficiency of C. albicans. The ura3 mutant CAI4
was transformed to Ura + with both circular and
linearized plasmid DNA of the URA3 integrative
vector, pJAM11. Pretreatment with LiAc/DTT/TE
resulted in an 3·5-fold elevation in the transformation frequency obtained with linearized DNA,
as shown in Table 3. Transformation efficiency
was also improved with circular DNA; however,
the precise fold-increase with circular DNA cannot
be calculated because in this case, no transformants were obtained in the absence of the LiAc/
DTT/TE incubation. Pretreatment with DTT
alone increased the transformation frequency obtained with linear DNA 2·5-fold but pretreatment alone with lithium acetate did not seem to
have an effect. However, on average, a combination of lithium acetate and DTT yielded higher
transformation frequencies with both linear and
circular DNA. Omission of the electroporation
step did not yield any transformants. Extending
the length of the LiAc/DTT/TE incubation to
overnight was detrimental and no transformants
were obtained.
All of the transformants grew when streaked on
selective media, indicating that they were stable
1998 John Wiley & Sons, Ltd.
integration events. Southern blot analysis was
performed to confirm that plasmid DNA had
been introduced into CAI4 by electroporation.
C. albicans is a diploid and the two alleles of the
URA3 locus can be distinguished by an EcoRI
restriction site polymorphism (Kelly et al., 1987).
A gel blot of EcoRI digests of genomic DNA
from strain CAI4 and nine transformants was
hybridized with a radiolabeled (32P) 1·5-kb XbaIScaI URA3 fragment isolated from pJAM11. As
shown in Figure 1, the probe did not hybridize to
DNA from parent strain CAI4, which has a
complete deletion of the 1·5-kb XbaI-ScaI fragment. In contrast, hybridization was obtained
with DNA from all of the Ura + transformants
tested. Each of the Ura + transformants contained
an 3·0-kb EcoRI fragment internal to pJAM11
and an additional EcoRI fragment of either
4·0 kb or 11·0 kb. The latter fragment sizes
are consistent with integration of pJAM11 into
either of the two alleles containing the URA3
We describe a modification of the electroporation
protocol of Becker and Guarente (1992). Our
protocol increases transformation efficiency in
S. cerevisiae strains approximately 1–2 orders of
. 14: 565–571 (1998)
    
Figure 1. Autoradiogram of Southern blot hybridization of
C. albicans Ura + transformants of CAI4. The lane containing
DNA from parent strain CAI4 is indicated. Ura + transformants 1 through 8 were obtained from transformation with
HpaI-digested pJAM11, and transformant 9 was obtained with
circular pJAM11. Lambda DNA digested with HindIII was
included as molecular size standards.
magnitude. We attribute the increase in efficiency
to pretreatment of the cells with LiAc/DTT/TE
and show that, for S. cerevisiae, both DTT and
LiAc contribute to the increased efficiency.
Various transformation protocols produce good
results with most Saccharomyces strains. However,
the additional step included in our procedure
employing a poorly transforming strain and/or in
situations such as library screening where maximal
transformation frequency is desirable.
Lithium acetate and DTT probably act to
enhance pore formation and the subsequent
uptake of DNA by directly affecting cell wall
structure. Either DTT or 2-mercaptoethanol alone
has been shown to release mannoproteins and
increase cell wall porosity (Brzobohaty and Kovac,
1986; DeNobel et al., 1989; Zlotnick et al., 1984).
DTT has been previously shown to enhance nonelectroporative transformation of LiAc competent
cells (Chen et al., 1992; Reddy and Maley, 1993).
DTT treatment alone has also been shown to
enhance electroporative transformation (Meilhoc
et al., 1990). Our work demonstrates that the
combination of LiAc and DTT has a roughly
multiplicative effect on electroporative transformation efficiency in S. cerevisiae. This may indicate
that LiAc and DTT affect different aspects of the
barrier to DNA entry into cells.
C. albicans transformation showed similar but
more modest benefits from treatment with LiAc
1998 John Wiley & Sons, Ltd.
and DTT. The cell walls of S. cerevisiae and
C. albicans have comparable but not identical
compositions (Tkacz, 1992) and we found that the
LiAc/DTT/TE pretreatment was not quite as
effective for C. albicans as it was for S. cerevisiae.
With C. albicans the LiAc plus DTT treatment
consistently showed a several-fold increase in
transformation efficiency over no pretreatment.
However, unlike S. cerevisiae, pretreatment with
LiAc alone did not enhance transformation of
C. albicans. Further, while the combination of
LiAc plus DTT, on average, produced the most
C. albicans transformants, the difference between
the efficiency of LiAc plus DTT and DTT alone is
not statistically significant. Thus, for C. albicans,
the DTT treatment appears to make a greater
contribution to the increase in transformation
effiency using this protocol. Cell wall integrity did
not appear to be dramatically compromised by the
concentrations of LiAc and DTT employed, as no
enhancement of the transformation frequency was
obtained by selection of any of the transformants
on synthetic medium containing 1 -sorbitol as
osmotic stabilizer for either S. cerevisiae or
C. albicans (data not shown).
Higher transformation frequencies were
achieved for C. albicans with linear DNA by our
electroporation procedure than we have obtained
with protocols requiring either treatment with
LiAc alone (Elbe, 1992) or digestion of the cell wall
to produce spheroplasts (Beggs, 1978). Our average electroporative transformation frequency was
254 transformants/ìg DNA with linearized
pJAM11 DNA (this value includes results from
additional experiments with LiAc/DTT-pretreated
cells) compared to 41 and 62/ìg for LiAc and
spheroplasting methods, respectively. While this
manuscript was in preparation, Brown et al. (1996)
described a different electroporation protocol for
C. albicans that yielded 0·7 transformants/ìg circular DNA with the Ura + plasmid pDBV53. This
is lower than the average value of 3·0/ìg that we
obtained with circular pJAM11 DNA. Perhaps
this is due in part to the fact that targeted
integration may not be as efficient with pDBV53
because the URA3 locus is slightly less homologous to the genome of their transformation host
than pJAM11 is for strain CAI4. Nevertheless,
our protocol has the advantage that electrocompetent cells do not have to be frozen for 24 h
to yield the reported transformation frequencies.
We expect that LiAc/DTT/TE pretreatment
will be useful in improving the electroporation
. 14: 565–571 (1998)
transformation efficiency of other Candida species
as well as other yeasts.
We are grateful to Cam Douglas, Mike Justice
and Steve Parent for critically reading the
Becker, D. M. and Guarente, L. (1991). High-efficiency
transformation of yeast by electroporation. Methods
Enzymol. 194, 182–187.
Becker, D. M. and Guarente, L. (1992). Protocol for
high-efficiency yeast transformation. In Chang, D. C.,
Chassy, B. M., Saunders, J. A. and Sowers, A. E.
(Eds), Guide to Electroporation and Electrofusion.
Academic Press, Inc., New York, pp. 31/501–31/505.
Beggs, J. D. (1978). Transformation of yeast by a
replicating hybrid plasmid. Nature (London) 275,
Brown Jr, D. H., Slobodkin, I. V. and Kumamoto, C. A.
(1996). Stable transformation and regulated expression of an inducible reporter construct in Candida
integration. Mol. Gen. Genet. 251, 75–80.
Brzobohaty, B. and Kovac, L. (1986). Factors enhancing genetic transformation of intact yeast cells modify
cell wall porosity. J. Gen. Micro. 132, 3089–3093.
Cannon, R., Jenkinson, H. F. and Shepard, M. G.
(1990). Isolation and nucleotide sequence of autonomously replicating sequence (ARS) element functional
in Candida albicans and Saccharomyces cerevisiae.
Mol. Gen. Genet. 235, 453–457.
Chen, D., Yang, B. and Kuo, T. (1992). One-step
transformation of yeast in stationary phase. Curr.
Genet. 21, 83–84.
DeNobel, J., Dijkers, C., Hooijberg, E. and Klis, F.
(1989). Increased cell wall porosity in Saccharomyces
cerevisiae after treatment with dithiothreitol or
EDTA. J. Gen. Micro. 135, 2077–2084.
Elbe, R. (1992). A simple and efficient procedure for
transformation of yeasts. BioTechniques 13, 18–20.
Fonzi, W. A. and Irwin, M. Y. (1993). Isogenic strain
construction and gene mapping in Candida albicans.
Genetics 134, 717–728.
Gaber, R., Copple, D., Kennedy, B., Vidal, M. and
Bard, M. (1989). The yeast gene ERG6 is required for
normal membrane function but is not essential for
biosynthesis of the cell-cycle-sparking sterol. Mol
Cell. Biol. 9, 3447–3456.
Gietz, R. and Sugino, A. (1988). New yeast–Escherichia
coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction
sites. Gene 74, 527–534.
1998 John Wiley & Sons, Ltd.
. .   .
Gillum, A. M., Tsay, E. Y. H. and Kirsch, D. R. (1984).
Isolation of the Candida albicans gene for orotidine5-phosphate decarboxylase by complementation of S.
cerevisiae ura3 E. coli pyrF mutations. Mol. Gen.
Genet. 198, 179–182.
Herreros, E., Garcia-Saez, M. I., Nombela, C. and
Sanchez, M. (1992). A reorganized Candida albicans
DNA sequence promoting homologous nonintegrative genetic transformation. Mol. Microbiol. 6,
Hill, J., Donald, K. A. G. and Griffiths, D. E. (1991).
DMSO-enhanced whole yeast cell transformation.
Nucl. Acids Res. 19, 5791.
Hoffman, C. S. and Winston, F. (1987). A ten-minute
DNA preparation from yeast efficiently releases
autonomous plasmid for transformation of Escherichia coli. Gene 57, 267–272.
Ito, H., Murata, K. and Kimura, A. (1984). Transformation of intact yeast cells treated with alkali cations
or thiol compounds. J. Biol. Chem. 48, 341–347.
Kelly, R., Miller, S. M., Kurtz, M. B. and Kirsch, D. R.
(1987). Directed mutagenesis in Candida albicans:
One-step gene disruption to isolate ura3 mutants.
Mol. Cell. Biol. 7, 199–207.
Kurtz, M. B., Cortelyou, M. W., Miller, S. M., Lai, M.
and Kirsch, D. R. (1987). Development of autonomously replicating plasmids for Candida albicans.
Mol. Cell. Biol. 7, 209–217.
Kurtz, M. and Scherer, S. (1991). Molecular genetics
of human fungal pathogens. In Bennett, J. and
Lasure, L. (Eds), More Gene Manipulations in Fungi.
Academic Press, Inc., New York, pp. 342–363.
Meilhoc, E., Masson, J. M. and Teissie, J. (1990). High
efficiency transformation of intact yeast cells by
electric field pulses. Biotechnology 8, 223–227.
Payne, G., Baker, D., vanTuinen, E. and Schekman, R.
(1988). Protein transport to the vacuole and receptormediated endocytosis by clathrin heavy chaindeficient yeast. J. Cell Biol. 106, 1453–1461.
Reddy, A. and Maley, F. (1993). Dithiothreitol
improves the efficiency of yeast transformation. Anal.
Biochem. 208, 211–212.
Rosenbluth, A., Mevarech, M., Koltin, Y. and Gorman,
J. A. (1985). Isolation of genes from Candida albicans
by complementation in Saccharomyces cerevisiae.
Mol. Gen. Genet. 200, 500–502.
Schiestl, R. H. and Gietz, R. D. (1989). High efficiency
transformation of intact yeast cells using singlestranded nucleic acids as carrier. Curr. Genet. 16,
Sherman, F., Fink, G. R. and Hicks, J. B. (1986).
Methods in Yeast Genetics: A Laboratory Course
Manual. Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY.
Sikorski, R. and Hieter, P. (1989). A system of shuttle
vectors and yeast host strains designed for efficient
manipulation of DNA in Saccharomyces cerevisiae.
Genetics 122, 19–27.
. 14: 565–571 (1998)
    
Thomas, B. and Rothstein, R. (1989). Elevated recombination rates in transcriptionally active DNA. Cell
56, 619–630.
Tkacz, J. S. (1992). Inhibition of cell wall glucan biosynthesis in fungi by papulacandin and echinocandin
antibiotics. In Sutcliffe, J. A. and Georgopapadakou,
N. (Eds), Emerging Targets for Antibacterial and
1998 John Wiley & Sons, Ltd.
Antifungal Therapy. Chapman and Hall, London,
pp. 495–523.
Zlotnik, H., Fernandez, M. P., Bowers, B. and Cabib, E.
(1984). Saccharomyces cerevisiae mannoproteins
form an external cell wall layer that determines wall
porosity. J. Bact. 159, 1018–1026.
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