All general growth conditions and yeast manipulations were performed as previously described (Moreno et al., 1991 (link); Forsburg and Rhind, 2006 (link)). The relevant genotypes and the source of the utilized strains are listed in Supplementary Table 1 . All experiments were performed with cells from cultures growing exponentially. Normally, cells were cultured in YES (Yeast Extract with Supplements; 0.5% yeast extract, 3% glucose, 225 mg/l adenine sulfate, histidine, leucine, uracil, and lysine). When required, cells were grown in EMM2 (Edinburgh Minimal Medium) (Moreno et al., 1991 (link)) with supplements. For drop-test analyses to assess sensitivity to ionic stress, 3 × 104 cells and serial 1:4 dilutions were inoculated on YES plates supplemented or not with different salts and incubated at 32°C for 3 days. When the assays were performed on minimal EMM2 plates, KNO3 was preferred to KCl for technical reasons, and the plates were incubated for 5 days. Geneticin (G418, Formedium), hygromycin B (Formedium), and nourseothricin (Werner BioAgents) were used at 120, 400, and 50 μg/ml, respectively.
Geneticin g418
Manufactured by Formedium
Geneticin (G418) is a broad-spectrum antibiotic commonly used as a selectable marker in cell culture and gene transfection experiments. It functions by inhibiting protein synthesis, thereby killing non-resistant cells.
Automatically generated - may contain errors
4 protocols using geneticin g418
1
Yeast Experimental Conditions Protocol
2
Yeast Cell Culture Conditions
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Yeast cells were cultured in either rich media (yeast extract peptone dextrose [YPD]; 2% glucose, 2% peptone, 1% yeast extract) or synthetic complete minimal medium (SC; 2% glucose, yeast nitrogen base supplemented with amino acid/base dropout mixtures). Cultures were routinely prepared in serial dilution overnight so that they were harvested for downstream experiments from early/mid–log phase (OD600 = <1.0). For glucose starvation experiments, 2% glucose media was washed 3× and exchanged with either identical media lacking any carbon source (no sugar) or media of the same recipe but instead containing 2% raffinose instead of glucose. KanMX and ClonNAT strain selections were performed in rich media containing 250 μg/ml geneticin/G418 (Formedium) or 150 μg/ml Nourseothricin (Jena Bioscience), respectively. GFP and mCherry fusions of SEC7 were created with a methotrexate cassette selected on 20 mM methotrexate (Alfa Aesar) supplemented with 5 mg/ml sulfanilamide. The loxP flanked cassette was then excised by TEF1-Cre expression and plasmid removal, as described (MacDonald and Piper, 2015 (link)). Expression of plasmids from the CUP1 promoter in appropriate selective media was induced by the addition of copper chloride (typically 20–100 µM).
3
Yeast Strain Handling and Growth Conditions
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All general growth conditions and yeast manipulations were performed as previously described [95 (link),96 (link)]. The relevant genotypes and the source of the utilized strains are listed in the S1 Table . Unless otherwise stated, the experiments were performed with cells from cultures growing exponentially in liquid rich medium, yeast extract with supplements (YES; 0.5% yeast extract, 3% glucose, 225 mg/l adenine sulfate, histidine, leucine, uracil and lysine, and 2% agar). Geneticin (G418, Formedium), hygromycin (Formedium), and nourseothricin (Werner BioAgents) were used at 120 μg/ml, 400 μg/ml, and 50 μg/ml, respectively. Edimburg minimal medium with 20 mM glutamate (a good nitrogen source; EMMG) or 20 mM phenylalanine (a poor nitrogen source; MMPhe) instead of 93.5 mM NH4Cl (EMM2) were used to analyze cell growth of prototrophic strains under nitrogen limiting conditions [49 (link),50 ]. Latrunculin A (Sigma; stock at 5 mM in dimethyl sulfoxide [DMSO]) was used at 100 μM for 20 minutes.
4
Yeast Cell Culture and Manipulation
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Yeast cells were cultured in either rich media (yeast extract peptone dextrose (YPD); 2% glucose, 2% peptone, 1% yeast extract) or synthetic complete minimal medium (SC; 2% glucose, yeast nitrogen base supplemented with amino acid / base dropout mixtures. Cultures were routinely prepared in serial dilution overnight so that cells were harvested for downstream experiments from early / mid-log phase log phase (OD600 = <1.0). For glucose starvation experiments, 2% glucose media was washed 3x and exchanged with either identical media lacking any carbon source (no sugar) or media of the same receipe but instead containing 2% raffinose instead of glucose. KanMX and ClonNAT strain selections were performed in rich media containing 250 μg/ml geneticin/G418 (Formedium) or 150 μg/ml Nourseothricin (Jena Biosceince), respectively. GFP and mCherry fusions of SEC7 were created with a methotrexate cassette selected on 20 mM methotrexate (Alfa Aesar) supplemented with 5 mg/ml sulphanilamide. The loxP flanked cassette was then excised by TEF1-Cre expression and plasmid removal, as described (MacDonald and Piper, 2015) . Expression of plasmids from the CUP1 promoter in appropriate selective media was induced by the addition of Copper chloride (typically 20 -100 µM).
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