Team:Caltech/Protocols/LacZ Assay

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# Grow cells overnight in 5 mL culture of LB Media with proper antibiotic at 37˚C and 225 RPM.  
# Grow cells overnight in 5 mL culture of LB Media with proper antibiotic at 37˚C and 225 RPM.  
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# 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 ul in a 96-well plate measured in a plate reader (Spectramax M2).
+
# 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.
# Combine 20 µL of cell culture and 380 µL permeabilization solution, mix, and place at 37˚C for 10 minutes.
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#* 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.
+
#* 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.
# 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.
#* 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.
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# When a faint yellow color is observed, add 300ul 1M sodium carbonate to stop the solution.
+
# 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>)).
# 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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LacZ Assay


  1. Grow cells overnight in 5 mL culture of LB Media with proper antibiotic at 37˚C and 225 RPM.
  2. 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).
  3. 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.
  4. 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.
  5. When a faint yellow color is observed, add 300 µl 1M sodium carbonate to stop the reaction.
  6. Miller units are calculated using the following equation: MU = 1000 (ABS420 / (0.02 * t * ABS600)).
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