Adapted from Current Protocols in Molecular Biology
- Grow 5ml YEPD cultures to mid-log phase.
- Centrifuge and resuspend cells in 5ml Z-buffer, then place on ice.
- Measure OD600.
- Use straight, or dilute cell mix 10x or 20x (40 or 80µl brought to 0.8mL
- Using Pasteur pipet, add 1 drop of 0.1% SDS and 2 drops of chloroform to
- Vortex well for 15 sec.
- Equilibrate @ 30° C for 15 min.
- Add 160µl of 4mg/ml ONPG, and vortex well for 10 sec.
- Incubate at 30°C and begin timing.
- Remove after about 15-20 min (empirically determined by color).
- Quench reaction by adding 400µl of 1M Sodium Carbonate.
- Spin down cell debris.
- Measure OD420 and OD550.
- Calculate Units using the following formula:
U= 1000 x [(OD420)-(1.75 x OD550)] / [(Time) x (Vol) x OD600]
Where Vol is volume of culture used in assay in mls, and Time is minutes at
DO NOT AUTOCLAVE
4mg/ml in 0.1M potassium phosphate buffer, pH 7, filter sterilized and stored
To make 0.1M potassium phosphate buffer, pH 7, you first need to make two solutions:
- Solution A: 27.2 g KH2PO4 in 1 L water.
- Solution B: 34.8 g K2HPO4 in 1 L water.
- Mix 39 ml Solution A and 61 ml Solution B and then add 100 ml of water.