Nov 17, 2025
SD 5-Foa Plates For Yeast Counter Selection
- Ryan Tan1
- 1NTU
Protocol Citation: Ryan Tan 2025. SD 5-Foa Plates For Yeast Counter Selection. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmbw7bg3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 18, 2025
Last Modified: November 17, 2025
Protocol Integer ID: 227560
Keywords: foa plates for yeast counter selection, yeast counter selection, foa plate, yeast, counter selection of ura3, prepartation of sd, sd, counter selection
Abstract
This protocol is for the prepartation of SD 5-Foa plates to use for the counter selection of URA3 in yeast.
Materials
6.7g/L Yeast Nitrogen Base (No amino acids, no amonium sulfate)
5g/L Ammonium Sulfate
2% (w/v) Glucose
2% (w/v) Agar
1g/L 5-Fluoroorotic Acid
50mg/L Uracil
(According to Manfucaturers specifications) -Ura Drop out Mix
Troubleshooting
Before start
Integrated marker cassette of ‘loxp-kanMX-URA-loxp’ is proven to be removed by the low-occurrence event of mitotic recombination between the repeated loxp-loxp, and positive colonies could be selected out by marker loss upon URA counter-selection in a one-step process.
Preparation of SD 5-Foa plates
In a clean bottle, add Yeast Nitrogen Base (No amino acids, no amonium sulfate), Ammonium Sulfate and glucose.
In another clean bottle, add Agar.
Add DI H2O to both bottles. (If preparing for 100mL, suggest to add 60mL to bottle containg Agar, and the rest of the 40mL to the other bottle)
Autoclave at 121oC for 15minutes.
Once cooled to around 50oC, add 5-Fluoroorotic Acid, Uracil, and -Ura Drop out Mix to the bottle containing Yeast Nitrogen Base (No amino acids, no amonium sulfate), Ammonium Sulfate and glucose.
Filter Sterile using 0.22um filter of contents in above bottle, into bottle containing Agar.
Dispense agar medium into petri dish (Recommend around ~15mL per petri dish)
1 day before experiment: 5mL culture of Cells
Innoculate transformed yeast cells from YPD agar plates (or whatever medium used) into 5mL of YPD. Remember to add markers in 5mL YPD.
Let it grow overnight in 30oC incubator, with shaking of 200rpm.
Day of Experiment: Plating onto SD 5 Foa Agar Plates
Centrifuge overnight culture at 1,100 xG for 5minutes.
Remove supernatant and wash cells with 5mL of sterile DI water.
Repeat washing step once more.
After final washing step, resuspend cells in 1mL of DI water
Plate 150 - 200uL of cells onto SD 5-Foa agar plates. (try not to plate too much cells onto agar plate, if not it will be overcrowded and cells will not grow)
If counter selection is successful, colonies should appear after 2-3 days.
2-3 Days After the Experiment: Confirm Counter Selection/ Marker Excision
Inoculate colonies from SD 5Foa plate into 5mL YPD medium.
Let it grow overnight in 30oC incubator, with shaking of 200rpm.
Plate 100 - 150uL of cells onto YPD agar with markers.
If counter selection and marker excision is successful, there should not be any colonies seen on the plates.
Protocol references
DOI: 10.1038/ncomms12851