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LacZ Assay
- Grow cells overnight in 5 mL culture of LB Media with proper antibiotic at 37˚C and 225 RPM.
- In the morning, back-dilute overnight cultures 1/100 (50 µL into 5mL) and grow for 3 hours at 37˚ and 225 RPM. The OD should be between 0.09 and 0.20 for 200 µl in a 96-well plate measured in a plate reader (Spectramax M2).
- Combine 20 µL of cell culture and 380 µL permeabilization solution, mix, and place at 37˚C for 10 minutes.
- Permeabilization Solution: 100mM Na2HPO4, 20mM KCl, 2 mM MgSO4, 0.6 mg/mL CTAB, 0.4 mg/mL sodium deoxycholate, and 5.4 µl/mL ß-mercaptoethanol.
- Combine 100 µL of lysed cells with 600 µL substrate solution.
- Substrate Solution: 60 mM Na2PO4, 40 mM NaH2HPO4, 1 mg/mL ONPG, and 5.4 µL/mL ß-mercaptoethanol.
- When a faint yellow color is observed, add 300 µl 1M sodium carbonate to stop the reaction.
- Miller units are calculated using the following equation: MU = 1000 (ABS420 / (0.02 * t * ABS600)).
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