Lab Protocols/Yeast Estrogen Assay
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- Measure OD640nm of overnight yeast
- Take 3x seven 1.5ml tubes and add 900ul 100% acetone
- Label as 100mg, 10mg, 1mg, 100ug, 10ug, 1ug and 100ng and as either EE2, E2 or E1
- To the first tube of each series, add 100ul 1g/L EE2, E2 or E1
- Vortex, remove 100ul to second tube and continue down the dilution series
- Take Xul of each dilution and pipette in to 96 well plate where appropriate
- For Y+M+A and M+A control wells add 6ul 100% acetone
- 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
- Add 200ul 1mg/ml CPRG to growth medium
- Divide growth medium in to 2x 10ml aliquots
- To one aliquot add Yeast to have OD640 of 0.04 in 10ml (333ul)
- Add 200ul Assay medium with/without yeast to appropriate wells
- Cover with lid or breathable membrane
- Measure OD 540 and OD640nm on plate reader (protocols are set up already)
- Incubate 32C for 3 days