USTC/BetaGalactosidaseAssay
From 2007.igem.org
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# Adjust the pH to 7.0 | # Adjust the pH to 7.0 | ||
# Bring the buffer to 500mL | # Bring the buffer to 500mL | ||
- | # Store at | + | # Store at 4°C |
'''Z Buffer:''' | '''Z Buffer:''' | ||
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# Bring the buffer to 500mL | # Bring the buffer to 500mL | ||
- | # Store at | + | # Store at 4°C |
'''Chloroform''' | '''Chloroform''' | ||
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# 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. | # 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. | ||
# Use 100µL cell suspension to measure the OD<sub>600</sub> (blank against Z buffer) | # Use 100µL cell suspension to measure the OD<sub>600</sub> (blank against Z buffer) | ||
- | # Permeabilize the diluted cells by adding 50µl chloroform and 25µl 0.1% SDS. Vortex (each sample exactly alike, e.g. 30 sec) | + | # 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. |
# Start reaction by adding 100µL 4mg/mL ONPG solution equilibrated to 28°C. Vortex and record the time of addition precisely with timer. | # Start reaction by adding 100µL 4mg/mL ONPG solution equilibrated to 28°C. Vortex and record the time of addition precisely with timer. | ||
# Incubate the cells at 28 C | # Incubate the cells at 28 C | ||
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* "Sufficient yellow color" means A420 should range from 0.6 to 0.9. Samples should be as yellow as unused LB broth. | * "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 [(OD<sub>420</sub> - 1.75 x OD<sub>550</sub>)] / (T x V x OD<sub>600</sub>) | * Miller Units = 1000 x [(OD<sub>420</sub> - 1.75 x OD<sub>550</sub>)] / (T x V x OD<sub>600</sub>) | ||
+ | # T = time of the reaction in minutes | ||
+ | # V = volume of culture used in the assay in mLs (in this protocol, V = 0.5mL) |
Revision as of 14:01, 23 September 2007
Reagents
Z Buffer (1X 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)
- Bring to approximately 400mL with H2O, dissolve all the salts
- Adjust the pH to 7.0
- Bring the buffer to 500mL
- Store at 4°C
Z Buffer:
- 50mL 1X Z Buffer stock solution (without beta-mercaptoethanol)
- 135µL beta-mercaptoethanol
- Should be prepared freshly
ONPG Solution:
- 20mL 1X Z Buffer stock solution (without beta-mercaptoethanol)
- 80mg ONPG
- Should be prepared freshly
Stop Solution:
- Bring the buffer to 500mL
- Store at 4°C
Chloroform
0.1% SDS
Protocol
- Grow culture until OD600 ranges from 0.3 to 0.7
- 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.
- Use 100µL cell suspension to measure the OD600 (blank against Z buffer)
- 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.
- Start reaction by adding 100µL 4mg/mL ONPG solution equilibrated to 28°C. Vortex and record the time of addition precisely with timer.
- Incubate the cells at 28 C
- Stop the reaction after sufficient yellow color has developed by adding 250µL 1M Na2CO3. Vortex and record the time of addition precisely with timer.
- Record the optical density at 420 nm and at 550 nm for each sample
- 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)
- T = time of the reaction in minutes
- V = volume of culture used in the assay in mLs (in this protocol, V = 0.5mL)