From 2007.igem.org

Reagents

1X Z Buffer stock solution (without beta-mercaptoethanol):

• 10.74g Na₂HPO₄ • 12 H₂O (0.06M)
• 3.12g NaH₂PO₄ • 2 H₂O (0.04M)
• 0.372g KCl (0.01M)
• 0.123g MgSO₄ • 7 H₂O (0.001M)

1. Bring to approximately 400mL with H₂O, dissolve all the salts
2. Adjust the pH to 7.0
3. Bring the buffer to 500mL
4. Store at 4°C

Z Buffer:

• 50mL 1X Z Buffer stock solution (without beta-mercaptoethanol)
• 135µL beta-mercaptoethanol

1. Should be prepared freshly

ONPG Solution:

• 20mL 1X Z Buffer stock solution (without beta-mercaptoethanol)
• 80mg ONPG

1. Should be prepared freshly

Stop Solution:

1M Na₂CO₃

1. Bring the buffer to 500mL
2. Store at 4°C

Chloroform

0.1% SDS

Protocol

1. Grow cultures until OD₆₀₀ ranges from 0.3 to 0.7. Incubate cultures 20min on ice to stop growth
2. Pellet 600µL cells at 4 C by centrifuging 10min at 6,000 rpm. Pour off the supernatant and resuspend the cell pellet in 600µL chilled Z buffer.
3. Use 100µL cell suspension to measure the OD₆₀₀ (blank against Z buffer)
4. Permeabilize the diluted cells by adding 50µl chloroform and 25µl 0.1% SDS. Vortex (each sample exactly alike, e.g. 30 sec) and equilibrate the samples 5min in a 28°C water bath.
5. Start reaction by adding 100µL 4mg/mL ONPG solution equilibrated to 28°C. Vortex and record the time of addition precisely with timer.
6. Incubate the cells at 28 C
7. Stop the reaction after sufficient yellow color has developed by adding 250µL 1M Na₂CO₃. Vortex and record the time of addition precisely with timer.
8. Record the optical density at 420 nm and at 550 nm for each sample
9. Calculate the units of activity

Comments

• "Sufficient yellow color" means A420 should range from 0.6 to 0.9. Samples should be as yellow as unused LB broth.
• Miller Units = 1000 x [(OD₄₂₀ - 1.75 x OD₅₅₀)] / (T x V x OD₆₀₀)

1. T = time of the reaction in minutes
2. V = volume of culture used in the assay in mLs (in this protocol, V = 0.5mL)