Difference between revisions of "5-Fluoroorotic Acid (5-FOA)"
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| − | 5-FOA (C<sub>5</sub>H<sub>3</sub>FN<sub>2</sub>O) counter selects for ura+ strains. The non-toxic 5-FOA is converted into toxic 5-flurouracil in yeast with a functional [https://www.pombase.org/gene/SPCC330.05c ''ura4''] gene | + | 5-FOA (C<sub>5</sub>H<sub>3</sub>FN<sub>2</sub>O) counter selects for ura+ strains. The non-toxic 5-FOA is converted into toxic 5-flurouracil (which blocks DNA replication) in yeast cells with a functional [https://www.pombase.org/gene/SPCC330.05c ''ura4''] gene that normally synthesizes uracil. We can thus select for strains that are ura-.<BR> |
* Make a 100X stock solution of 5-FOA in DMSO by adding 100 mg/ml. <BR> | * Make a 100X stock solution of 5-FOA in DMSO by adding 100 mg/ml. <BR> | ||
* For use, use 1:100 (200 µl/9cm plate or 3µl/300µl in Deep Well Plate).<BR> | * For use, use 1:100 (200 µl/9cm plate or 3µl/300µl in Deep Well Plate).<BR> | ||
* Stock solution is available in freezer 7.<BR> | * Stock solution is available in freezer 7.<BR> | ||
* Keep plates in fridge. | * Keep plates in fridge. | ||
| − | + | The method works with both [[PMG - Pombe Minimal Glutamate|minimal medium]] (with reduced amount of uracil) or [[YES - Yeast Extract Supplemented|YES]], however according to a discussion on PombeList (16 Sept 2019) works better with rich medium. For a good protocol on how to use this with YES, see: [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5526333/ Petersen & Russell] | |
| − | For a good protocol on how to use this with YES, see: [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5526333/ Petersen & Russell] | ||
| − | |||
[[Category:Media]] | [[Category:Media]] | ||
[[Category:Stock Solutions]] | [[Category:Stock Solutions]] | ||
Latest revision as of 17:05, 21 June 2022
5-FOA (C5H3FN2O) counter selects for ura+ strains. The non-toxic 5-FOA is converted into toxic 5-flurouracil (which blocks DNA replication) in yeast cells with a functional ura4 gene that normally synthesizes uracil. We can thus select for strains that are ura-.
- Make a 100X stock solution of 5-FOA in DMSO by adding 100 mg/ml.
- For use, use 1:100 (200 µl/9cm plate or 3µl/300µl in Deep Well Plate).
- Stock solution is available in freezer 7.
- Keep plates in fridge.
The method works with both minimal medium (with reduced amount of uracil) or YES, however according to a discussion on PombeList (16 Sept 2019) works better with rich medium. For a good protocol on how to use this with YES, see: Petersen & Russell
