Team:Caltech/Protocols/LacZ Assay

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Latest revision as of 00:43, 30 October 2008

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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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+# 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 Na<sub>2</sub>HPO<sub>4</sub>, 20mM KCl, 2 mM MgSO<sub>4</sub>, 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 NaH<sub>2</sub>HPO<sub>4</sub>, 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 (ABS<sub>420</sub> / (0.02 * t * ABS<sub>600</sub>)).
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