PMC:4502370 / 14459-15753 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4502370","sourcedb":"PMC","sourceid":"4502370","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4502370","text":"Plasmid shuffle assay for rpo26Δ complementation\nA heterozygous diploid S. cerevisiae RPO26rpo26::kanMX heterozygous strain (purchased from Open Biosystems) was transformed with pRS316-RPO26. The resulting Ura+ diploid was sporulated and tetrads were dissected. We thereby recovered viable rpo26::kanMX haploids that were resistant to G418 and unable to grow on medium containing 0.75 mg/ml FOA (5-fluoroorotic acid), a drug that selects against the URA3RPO26 plasmid. The rpo26Δ strain was used to test plasmid-borne alleles of RPO26 for rpo26Δ complementation by plasmid shuffle as follows. rpo26Δ pRS316-RPO26 cells were transfected with pRS425-TPI-RPO26 plasmids containing wild-type or mutant RPO26 alleles. Individual transformants were selected and patched on SD-Leu agar medium. Cells from each isolate were streaked on agar medium containing 1.0 mg/ml FOA at 30°. In cases where the RPO26-containing plasmid supported growth on FOA, two isolates of each mutant amplified from single FOA-resistant colonies were tested for growth by spotting 3-μl aliquots of serial 10-fold dilutions of cells (from liquid cultures grown in SD–Leu medium to mid-log phase at 30° and adjusted to A600 of 0.1) to YPD agar and incubating the plates at 18° for 7 d, 25° for 4 d, 30° for 3 d, or 37° for 2 d.","divisions":[{"label":"title","span":{"begin":0,"end":48}}],"tracks":[]}