A ten-minute protocol for transforming Saccharomyces cerevisiae by electroporation

  • Published on

  • View

  • Download

Embed Size (px)


<ul><li><p>Curr Genet (1992)22:335-336 Current Genetics 9 Springer-Verlag 1992 </p><p>Short communications </p><p>A ten-minute protocol for transforming Saccharomyces cerevisiae by electroporation Martin Grey and Martin Brendel </p><p>Institut fiir Mikrobiologie der J. W. Goethe-Universit~it, Theodor-Stern-Kai 7, Haus 75, W-6000 Frankfurt/Main, Federal Republic of Germany </p><p>Received April 24, 1992 </p><p>Summary. We present a simplified and rapid method for the transformation of yeast ceils by electroporation. Sta- tionary cells, scraped off the agar of Petri dish cultures stored in the refrigerator for up to 6 weeks, are suspended in sorbitol buffer, spun down by gentle centrifugation, transferred into the electroporation cuvette, and immedi- ately subjected to transformation via electroporation. Transformation efficiency of this 10-rain method, which does not require the preparation of cell cultures, is about 10 % of the hitherto best performing transformation pro- cedure using cells of defined growth phase. </p><p>Key words: Yeast - Rapid transformation - Cell age </p><p>Introduction </p><p>The use of high-voltage electric shocks has become a valuable technique for the introduction of exogenous DNA into prokaryotic and eukaryotic cells (Neumann etal. 1982; Wong and Neumann 1982; Fromm etal. 1985; Dower et al. 1988; Shigekawa and Dower 1988). Several procedures have been described to transform Saccharomyces cerevisiae by electroporation (Hashimoto et al. 1985; Karube et al. 1985; Delorme 1989). Since the main interest was focused on a high efficiency of transfor- mation many parameters had to be optimized. Conse- quently the evolved high efficiency electroporation pro- tocol (Becker and Guarente 1991) requires strict control of the growth phase of cells, which amounts to many time-consuming steps of cell handling before electropora- tion. </p><p>However, in most cases of yeast transformation (with the exception of screening a gene bank) high efficiency of transformation is not the prime goal of the experiment. Since in routine experimentation only one defined plas- mid or DNA fragment is to be transformed into yeast not a high number of identical transformants but simplicity </p><p>Correspondence to: M. Brendel </p><p>of the method is the main objective. With this in mind we have developed an extremely simple procedure that makes it possible to transform yeast cells with an accept- able efficiency within 10 min, counted from the inception of the experiment to the transfer of transformed and plat- ed cells to the incubator. </p><p>The ten-minute transformation protocol </p><p>Collect cells of the desired yeast strain (normally kept on agar medium in the refrigerator at 4~ using a sterile transfer loop and suspend them in an Eppendorf tube containing approximately 1 ml of electroporation-buffer (1 M sorbitol, 20 mM HEPES). Vortex briefly to ensure that clumps of yeast cells have dispersed. Spin down the cells at room temperature for 1 min at about 4000 rpm using a tabletop centrifuge. Aspirate the supernatant, re- suspend cells in the remaining liquid by stirring with the tip of the pipette, which is then used to transfer 40 gl of this suspension into a sterile electroporation cuvette. </p><p>Add 200 to 1000 ng of (plasmid) DNA dissolved in maximally 5 gl TE buffer and mix gently. Pulse at 1.4 kV, 200 ohms, 25 pF with a Gene Pulser using cuvettes with an 0.2 mm electrode-gap (Bio-Rad, Munich, FRG). Im- mediately add 1 ml 1 M sorbitol using a Pasteur pipette and mix gently to ensure a thorough suspension of the cells in sorbitol. Spread aliquots directly (i.e., without any further incubation) onto appropriate selective agar plates containing 1 M sorbitol. Incubate at 28 ~ to 30 ~ for 3 days. </p><p>With this protocol we have transformed two haploid yeast strains EH3714-2B (MATa, ura3-52, Ieu2-3,112, his5-2, ade2-1, lysl-1) and MG5123-6B (MATe, ura3-52, his5-2, ade2-1) with the two episomal plasmids YEp352 (Hill et al. 1986) and YEp24 (New England Biolabs, Schwalbach, FRG). The yeast cells used in the transfor- mation experiments came from agar-plate cultures stored in the refrigerator for up to 6 weeks. As can be seen from Fig. 1 (open symbols) the time of storage had some effect on the yield of transformants, with transformation effi- </p></li><li><p>336 </p><p>10 4 </p><p>&lt; z c3 </p><p>\ u) </p><p>g E 3 </p><p>10 3 </p><p>_1 I [ t I I I </p><p>I I I I I I 0 2 L 6 </p><p>age of culture [weeks) </p><p>Fig. 1. Transformation efficiency with YEp352 of stationary hap- loid yeast strain EH3714-2B in relation to storage time of yeast culture at 4 ~ Open squares and triangles, electroporation in the presence and absence of 20 mM HEPES, respectively. Filled sym- bols, number of transformants corrected for viability of cells before electroporation. At zero weeks age the cells had been growing on solid medium for 3 days at 30 ~ </p><p>ciency decreasing by a factor of approximately two after 6 weeks storage at 4 ~ of the yeast cultures. This is main- ly due to the fact that survival in the refrigerated agar cultures decreases by a factor of approximately 1.6. Sur- vival of yeast cells after the electroporation process itself was found to be constant at about 50%, regardless of age. When sorbitol was buffered by 20 m M HEPES the trans- format ion efficiency increased by a factor of 2.5 irrespec- tive of the age of the yeast cells (Fig. 1). Transformation efficiency was similar for both yeast strains and the size of the plasmids had no significant influence (Fig. 2), a fact already reported by Becker and Guarente (1991). Also, as shown by these authors and by Delorme (1989), carrier D N A did not increase t ransformation efficiency (data not shown). When the above-mentioned strains were t ransformed with YEp352 according to the protocol described by Becker and Guarente (1991) t ransformant yield was about ten-fold higher. </p><p>We hope that this 10-rain electroporation protocol, which emphasises rapidity in execution rather than max- imal t ransformation efficiency, may contribute to the flexibility in the daily lab routine of molecular geneticists. </p><p>Acknowledgements. We thank Ms. M. Markovic for drawing the figures and Dr. E. Haase for the yeast strain. </p><p>~-~ 10' </p><p>&lt; 8' Z </p><p>xz6. </p><p>gt. . E </p><p>~ 2 g </p><p>0 I 6 oge of culture (weeks) </p><p>Fig. 2. Transformation efficiency of two haploid yeast strains in stationary growth phase in relation to storage time at 4 ~ Strains EH3714-2B (first two columns in each group) and MG5123-6B were transformed with multi-copy plasmids YEp24 (open and stippled) and YEp352 (hatched andsolid), respectively. Data not corrected for cell viability </p><p>References </p><p>Becker DM, Guarente L (1991) Methods Enzymol 194:182-187 Delorme E (1989) Appl Environ Microbiol 55:2242-2246 Dower WJ, Miller JF, Ragsdale CW (1988) Nucleic Acids Res </p><p>16:6127 6145 Fromm M, Taylor LP, Walbot V (1985) Proc Natl Acad Sci USA </p><p>82: 5824- 5828 Hashimoto H, Morikawa H, Yamada Y, Kimura A (1985) Appl </p><p>Microbiol Biotechnol 21:336-339 Hill JE, Myers AM, Koerner TJ, Tzagoloff A (1986) Yeast 2:163- </p><p>167 Karube I, Tamiya E, Matsuoka H (1985) FEBS Lett 182:90-94 Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH (1982) </p><p>EMBO J 1:841-845 Shigekawa K, Dower WJ (1988) BioTechniques 6:742-751 Wong TK, Neumann E (1982) Biochem Biophys Res Commun </p><p>107:584-587 </p><p>Communicated by K. Wolf </p></li></ul>


View more >