Large-scale yeast transformation in low-percentage agarose medium

BENCHMARKS The use of yeast as a research tool became especially popular with the development of the yeast two-hybrid (Y2H) assay, which is a powerful tool for the detection of specific protein-protein interactions. Several versions of the Y2H system have been developed. All variations of this method demand the large-scale transformation of yeast with cDNA library plasmids (1). Transformation of yeast with plas-mid DNA can be achieved by several methods, such as agitation with glass beads (2), spheroplast preparation (3), treatment with lithium acetate (4), or electroporation (5). The most popular and commonly used is lithium acetate transformation was developed by Ito et al. (6) and modified by Gietz and Woods (7). The standard Y2H library protocols for an average mammalian cDNA library (10 6 primary independent clones) and maximal colony density 5/mm 2 require plating of more than fifty 150-mm plates (8). In addition to being laborious and having problems in maintaining sterility, the inevitable overgrowth of marginal colonies near the plate edge causes loss of uniformity across the library. Here we present a protocol for the large-scale transformation of yeast in a semi-solid agarose medium that does not require plating. The protocol allows for the growth of transformed cells as separate colonies in the volume of semi-solid medium. The protocol is optimized for lithium acetate transformation , gives high transformation efficiency , and provides an equal growth environment for each colony, thereby faithfully preserving the integrity of the primary library. It also allows yeast library propagation with a significantly higher colony density. The protocol was developed on the basis of several published techniques (7–10) and requires fewer reagents when compared to other approaches. Working with DupLEX-A™, a LexA-based Y2H system (OriGene Technologies, Rockville, MD, USA), we successfully used this protocol for the EGY48 strain (MATα, his3, trp1, ura3, 6 LexAop-LEU2) transformed with different pEG202-based bait plas-mids and a reporter plasmid pSH18-34 (10). Using this protocol, we managed to achieve transformation efficiencies of 0.25–1 × 10 6 /µg of library DNA or 2–8 million independent colonies/400 mL of the semi-solid agarose medium. Therefore, this protocol allowed us to cover an average cDNA library with just one or two bottles of semi-solid medium (400 mL each). All reagents used were from Sigma (St. Louis, MO, USA) except the low melting point agarose). Culture media reagents as well as carrier DNA were from Bio101 (Vista, CA, USA). Procedures were performed according …