Lab Protocols/Yeast Estrogen Assay

1. Measure OD640nm of overnight yeast
2. Take 3x seven 1.5ml tubes and add 900ul 100% acetone
3. Label as 100mg, 10mg, 1mg, 100ug, 10ug, 1ug and 100ng and as either EE2, E2 or E1
4. To the first tube of each series, add 100ul 1g/L EE2, E2 or E1
5. Vortex, remove 100ul to second tube and continue down the dilution series
6. Take Xul of each dilution and pipette in to 96 well plate where appropriate
7. For Y+M+A and M+A control wells add 6ul 100% acetone
8. Make up 20ml growth medium:

• • 20% D-Glucose - 2ml
  • L-aspartic acid - 0.5ml
  • Vitamin solution - 0.2ml
  • L-Threonine - 0.4ml
  • Copper II Sulphate - 50ul
  • Minimal media - 18ml

1. Add 200ul 1mg/ml CPRG to growth medium
2. Divide growth medium in to 2x 10ml aliquots
3. To one aliquot add Yeast to have OD640 of 0.04 in 10ml (333ul)
4. Add 200ul Assay medium with/without yeast to appropriate wells
5. Cover with lid or breathable membrane
6. Measure OD 540 and OD640nm on plate reader (protocols are set up already)
7. Incubate 32C for 3 days