Encyclopedia of Experiments: Biological Techniques
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Resuspend the culture, and transfer 125 microliters from each well to a spectrophotometer plate. Measure the optical density at 600 nanometers to ensure it is between 0.3 and 0.6. Centrifuge the remaining cells in the deep well block at 3,000 times g and 21 degrees Celsius for 10 minutes. Remove the supernatant by inverting.
Add 200 microliters of Z-buffer to each well and vortex. Centrifuge at 3,000 times g and 21 degrees Celsius for 5 minutes. Remove the supernatant by inverting. Add 20 microliters of Z-buffer to each well and vortex. Cover the plate with sealing foil that is resistant to freeze-thaw cycles.
In a fume hood, perform four cycles of freeze-thaw using liquid nitrogen in a 42-degree Celsius water bath. Add 200 microliters of freshly-prepared Z-buffer/beta-mercaptoethanol/ONPG solution to each well. Incubate at 30 degrees Celsius for 17 to 24 hours or until color develops. Check that the color has developed.
Add 110 microliters of 1 molar sodium carbonate to each well to stop the reaction. Record the time, and vortex the plate. Centrifuge at 3,000 times g and 21 degrees Celsius for 10 minutes. Use a multichannel pipette to transfer 125 microliters of supernatant from each well to a spectrophotometer plate. Measure the optical density at 420 nanometers.
