Team:Caltech/Protocols/LacZ Assay

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LacZ Assay

ß-galactosidase assays were performed as follows. Cells were grown overnight in 5 mL culture of LB Media with proper antibiotic at 37˚C and 225 RPM. Overnight cultures were back-diluted 1/100 (50 µL into 5mL) and grown for 3 hours at 37˚ and 225 RPM. OD600 values were taken on the spectrophotometer. Cells were used at OD values between 0.09 and 0.20. 20 µL of our cell culture and 380 µL permeabilization solution (100mM Na2HPO4, 20mM KCl, 2 mM MgSO4, 0.6 mg/mL CTAB, 0.4 mg/mL sodium deoxycholate, and 5.4 ul/mL ß-mercaptoethanol) were mixed and placed at 37˚C for 10 minutes. 100 µL of lysed cells were then mixed with substrate solution (60 mM Na2PO4, 40 mM NaH2PO4, 1 mg/mL ONPG, and 5.4 µL/mL ß-mercaptoethanol). When a faint yellow color was observed, 300ul 1M sodium carbonate was added to stop the solution.
Miller units were calculated using the following equation: 1000 (ABS420 / (0.02 mL * t * ABS600).

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-ß-galactosidase assays were performed as follows. Cells were grown overnight in 5 mL culture of LB Media with proper antibiotic at 37˚C and 225 RPM. Overnight cultures were back-diluted 1/100 (50 µL into 5mL) and grown for 3 hours at 37˚ and 225 RPM. OD600 values were taken on the spectrophotometer. Cells were used at OD values between 0.09 and 0.20. 20 µL of our cell culture and 380 µL permeabilization solution (100mM Na2HPO4, 20mM KCl, 2 mM MgSO4, 0.6 mg/mL CTAB, 0.4 mg/mL sodium deoxycholate, and 5.4 ul/mL ß-mercaptoethanol) were mixed and placed at 37˚C for 10 minutes. 100 µL of lysed cells were then mixed with substrate solution (60 mM Na2PO4, 40 mM NaH2PO4, 1 mg/mL ONPG, and 5.4 µL/mL ß-mercaptoethanol). When a faint yellow color was observed, 300ul 1M sodium carbonate was added to stop the solution. Miller units were calculated using the following equation:
+ß-galactosidase assays were performed as follows. Cells were grown overnight in 5 mL culture of LB Media with proper antibiotic at 37˚C and 225 RPM. Overnight cultures were back-diluted 1/100 (50 µL into 5mL) and grown for 3 hours at 37˚ and 225 RPM. OD600 values were taken on the spectrophotometer. Cells were used at OD values between 0.09 and 0.20. 20 µL of our cell culture and 380 µL permeabilization solution (100mM Na2HPO4, 20mM KCl, 2 mM MgSO4, 0.6 mg/mL CTAB, 0.4 mg/mL sodium deoxycholate, and 5.4 ul/mL ß-mercaptoethanol) were mixed and placed at 37˚C for 10 minutes. 100 µL of lysed cells were then mixed with substrate solution (60 mM Na2PO4, 40 mM NaH2PO4, 1 mg/mL ONPG, and 5.4 µL/mL ß-mercaptoethanol). When a faint yellow color was observed, 300ul 1M sodium carbonate was added to stop the solution. <BR>Miller units were calculated using the following equation:
 (ABS420 / (0.02 mL * t * ABS600).
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