Cryopreserved yeast cells suitable for routine, small-scale transformations.

Yeast strains have been widely used as models for eukaryotic microorganisms because of their amenability to biochemical and genetic studies. Recent advances of molecular biological techniques have made this organism an attractive host for functional expression of heterologous genes. Furthermore, structural genes corresponding to various genetic traits have been identified by complementation studies in yeast with recombinant plasmid libraries of mammalian origin (12,13,15). The use of yeast as a research tool has also been popularized by the development of yeast two-hybrid system for studying protein-protein interactions (7). Introduction of plasmid DNA into yeast can be achieved by several methods, including: agitation with glass beads (3), spheroplast preparation (10), treatment with lithium acetate (LiAc) (8) and electroporation (1). These methods are laborious and time-consuming, particularly when multiple samples are involved. The recent availability of commercially prepared competent yeast cells and yeast transformation kits should help ease the problem. However for routine, small-scale transformations, the use of these products is not economical. Thus, several laboratories have reported variations of the LiAc protocol (8) for preparing competent yeast cells (5,9), including one method that enables long-term storage of competent yeast cells (4). Here we describe a simple and reliable procedure for preparing Saccharomyces cerevisiae cells that can be stored frozen and used as needed for small-scale transformations (Table 1). The protocol yields 100 to over 1000 S. cerevisiae transformants per μg of plasmid DNA without a significant decrease in transformation efficiencies, even when the cells have been cryopreserved up to 8 months (Table 2). We have used this method successfully to prepare competent S. cerevisiae strains Hf7c (MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901, leu2-3,112, gal4-542, gal80-538, LYS2::GAL1→HIS3, +URA3::(GAL4 17mers)3-CYC1→lacZ; Reference 6), a reporter yeast strain for the two-hybrid system (7) and RY1-1 (MATa, ura3-52 leu2-3,-112 gcn2∆trp1-∆63 LEU2:: GAL-CYC1-PKR)2; Reference 14). Media used to culture yeast strains were described previously (6,14). Plasmids pGBT9 and/or pGAD424 (CLONTECH Laboratories, Palo Alto, CA, USA) were purified from Escherichia coli strain DH5α (Invitrogen, Carlsbad, CA, USA) by the standard alkaline lysis method (2) and then used to transform Hf7c; whereas, plasmids pYES2 and/or pYX233 (Invitrogen) were transformed into RY1-1. Frozen competent yeast cells were thawed at room temperature, microcentrifuged (room temperature at 14 000 rpm for 5 s in a Model 5415C Centrifuge [Eppendorf, Madison, WI, USA]) and then resuspended in 0.1 mL freshly prepared TE/LiAc solution. Yeast transformations were carried out essentially as described by Gietz and Schiestl (8). One hundred nanograms of plasmid DNA (1 μg of each plasmid for dual transformations; ≤3 μL total volume) and 100 μg of denatured salmon sperm carrier DNA (Catalog No. 2200-205; Bio 101, Vista, CA, USA) were added to 0.1 mL of competent yeast cells, and vortex mixed thoroughly in the microcentrifuge tube. After incubation at room temperature for 5 min, 0.6 mL of sterile polyethylene glycol (PEG)/LiAc solution (40% PEG 4000 [Sigma Chemical, St. Louis, MO, USA], 100 mM LiAc, 1 mM EDTA, 10 mM Tris-HCl, pH 7.5) was added to this tube and vortex mixed thoroughly. Seventy microliters of dimethyl sulfoxide were added to the tube, and the solution was mixed gently. Cells were then (i) heat shocked at 42°C for 15 min with gentle shaking every 5 min, (ii) placed on ice for 2 min, (iii) microcentrifuged (room temperature at 14 000 rpm for 5 s in a Model 5415C Centrifuge) and (iv) washed with 500 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). The washing step removes PEG, which can have negative effects on cell growth. The cell pellet was then resuspended in 0.5 mL of TE buffer. Aliquots of 0.1 mL of the cell suspension were then spread on Synthetic Defined Dropout Media (Bio 101), and transformants appeared after 3 days at 30°C. The full number of transformants was recorded after the plates were incubated for 5 days. The transformation efficiency obtained from using our protocol is comparable to that achieved from using the protocol described by Gietz et al. (9). Our method should help alleviate the time-consuming and cumbersome Benchmarks