Team:Caltech/Protocols/LacZ Assay

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Revision as of 00:40, 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 to an OD between 0.09 and 0.20 as assessed by plate reader ().
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 ul/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 300ul 1M sodium carbonate to stop the solution.
Miller units are calculated using the following equation: MU = 1000 (ABS₄₂₀ / (0.02 * t * ABS₆₀₀)).

@@ Line 8: / Line 8: @@
 # 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 grown for 3 hours at 37˚ 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 to an OD between 0.09 and 0.20 as assessed by plate reader ().
-#* Regrow cells to OD values between 0.09 and 0.20.
 # 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 ul/mL ß-mercaptoethanol.