Lithium Acetate Transformation of PCR Products

CEN5 was deleted as previously described [32 (link)]. Lithium acetate transformation of PCR products with at least 70 bp of homology to the targeted gene was used for strain construction. Briefly, strains to be transformed were inoculated in liquid YPAD (10g/L yeast extract, 20g/L bactopeptone, 0.04g/L adenine, 0.08g/L uridine, 20g/L dextrose) and grown at 30°C for 16–18 h. Cultures were then diluted 1:166 in YPAD and grown at 30°C for 3–4 h. Cells were washed with water, then TELiAc (10mM Tris pH 7.5, 1mM EDTA, 100mM LiAc) and incubated in TELiAc with transformation DNA and 50μg sheared salmon sperm DNA (Ambion) for 30 min. 4 volumes PLATE mix (40% PEG, 10mM Tris pH 7.5, 1mM EDTA, 100mM LiAc) was then added and the transformation mix was incubated for 16–18 h at 20–24°C. Transformations were heat shocked at 42°C for 1 h, then plated on selective media with the exception of NAT1 marker transformations, which were recovered on non-selective media for 6 h prior to replica plating to selective media containing 400 μg/ml nourseothricin (Werner BioAgents). Strains were checked by PCR of genomic DNA.