Yeast cultures were grown according to standard procedures [36 ,37 ]. The parental strains, BY4741, BY4743, CEN.RO16, WDAM006, and α-D273 (genotype information available in S3 Table ), were maintained in rich YPD (Yeast Peptone Dextrose) media and grown in YP-galactose (YPGal) media as inductive conditions. Yeast strains carrying mutations in the endogenous FAE component genes were obtained from Open Biosystems (Huntsville, AL) and are listed in S3 Table . These strains and all derivatives that carry the gene-disrupting KanMX4 cassette were selected by growth on media containing 200 μg/mL Geneticin (G418; Invitrogen, Carlsbad, CA). Yeast strains carrying maize expression cassettes were selected by their ability to grow on minimal medium (SD) without the appropriate amino acid or nucleobase (e.g., uracil). Expression of maize FAE components was induced with the inclusion of 2% galactose in YPGal medium. Counter-selection of URA3 carrying vectors was done in the presence of 100 μg/mL 5-fluoroorotic acid (5-FOA; US Biological, Swampscott, MA) in SD media.
5 fluoroorotic acid 5 foa
Manufactured by US Biological
5-fluoroorotic acid (5-FOA) is a chemical compound used in molecular biology and genetics research. It serves as a selective agent, allowing for the identification and isolation of specific genetic mutations or cell lines. 5-FOA inhibits the growth of cells that can utilize the uracil biosynthesis pathway, making it a useful tool for researchers studying this cellular process.
Automatically generated - may contain errors
Lab products found in correlation
Geneticin g418, by Thermo Fisher Scientific (1 mentions)
Peptone, by Thermo Fisher Scientific (1 mentions)
Kanamycin, by Merck Group (1 mentions)
D camphor, by Merck Group (1 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
Doxycycline dox, by Takara Bio (1 mentions)
Carbenicillin, by Merck Group (1 mentions)
Geneticin, by Santa Cruz Biotechnology (1 mentions)
Hygromycin b, by Thermo Fisher Scientific (1 mentions)
5 foa, by US Biological (1 mentions)
4 protocols using 5 fluoroorotic acid 5 foa
1
Yeast Strain Cultivation and Selection
2
Yeast and E. coli Culture and Transformation
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Yeast cultures were grown in either YPD (1% yeast extract) US Biological Life Sciences, Swampscott, MA, USA, 2% glucose (VWR, Radnor, PA, USA), 2% peptone (Thermo Fisher Scientific, Waltham, MA, USA) or in SD (0.67% yeast nitrogen base without amino acids and carbohydrates (US Biological Life Sciences), 2% glucose), supplemented with the appropriate nutrients to select for plasmids and tagged genes. Escherichia coli DH5α was used to propagate all plasmids. E. coli cells were cultured in Luria broth medium (1% Bacto tryptone, 0.5% Bacto yeast extract, 1% NaCl) and transformed to ampicillin resistance by standard methods. Hsp70 isoform plasmids were transformed into yeast strain ssa1–4∆ [27 (link)] using PEG/lithium acetate. After restreaking onto media lacking leucine, transformants were streaked again onto media lacking leucine and containing 5-fluoro-orotic acid (5-FOA) (US Biological Life Sciences), resulting in yeast that expressed Hsp70 paralogs as the sole cytoplasmic Hsp70 in the cell. For a full description of yeast strains see Table 1 and for plasmids see Table 2 .
3
Yeast Cultivation and Strain Selection
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Yeast strains were cultured in YPD medium or SD-based media supplemented with appropriate amino acids; fully supplemented medium containing all amino acids plus uracil and adenine is referred to as SC. SC–Ade is SC lacking adenine. D-Camphor was purchased from Sigma-Aldrich (St. Louis, MO), and 5-fluoroorotic acid (5-FOA) was from US Biological (Massachusetts, MA). Doxycycline (Dox) was obtained from Clontech laboratories (Mountain View, CA). Escherichia coli was grown in Luria Broth (LB) media. To select strains with drug-resistant genes, carbenicillin (Sigma-Aldrich), kanamycin (Sigma-Aldrich), or zeocin (Life Technologies, Carlsbad, CA) were used at final concentrations of 75 µg/ml, 50 µg/ml, and 25 µg/ml, respectively. Agar was added to 2% for preparing solid media.
4
Yeast Strains and Cultivation Protocols
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Strains used in this study (Supplementary Table 6 ) are derivatives of the S288c strain RDKY596420 (link), with exception of strain AH109 (Clontech Laboratories) that was used for Y2H analysis. Strains were cultivated at 30 °C in yeast extract-peptone-dextrose media (YPD) or appropriate dextrose-containing synthetic dropout (SD) medium for selection of plasmids markers, lacking lysine (Lys−) or threonine (Thr−) (to select for lys2-10A or hom3-10 frameshift revertants, respectively), or SD medium lacking arginine (Arg−) supplemented with 60 mg/L canavanine, to select canavanine-resistant (CanR) mutants. 5-fluoroorotic acid (5-FOA, US Biological) plates were done in SD medium supplemented with 1 g/L 5-FOA. Antibiotics were used at the following final concentrations: 200 μg/mL geneticin (Santa Cruz Biotechnology), 300 μg/mL hygromycin B (Thermo Fisher Scientific), and 100 μg/mL nourseothricin (clonNAT, Werner BioAgents). Gene deletions and gene tagging were performed using standard PCR-based recombination methods51 (link),52 (link), followed by confirmation by PCR. Tags and junctions were confirmed by PCR and sequencing. Yeast strains carrying mutations in MLH1, MLH2, or PMS1 genes, were generated by pop-in/pop-out strategy using pRS306-based integrative vectors51 (link), and were confirmed by sequencing.
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