Genetics - β-Galactosidase Assays
Jeff Lawrence (G50)
1. Grow cells overnight in minimal media.
2. Inoculate 5 mL of assay media with 100 µL fresh overnight culture. Grow at 37° until culture reaches 5x108 cells/mL (OD600 of 0.7).
3. Inoculate 5 mL of assay media with 100 µL of this culture. Grow at 37° until culture reaches 2 - 5x108 cells/mL (OD600 of 0.2 - 0.7). This dilution ensures that cells are in mid-log phase
4. Chill culture on ice for 20 min. Record cell density by measuring OD600.
5. Prepare ZS-buffer as (add SDS solution afterwards for 1.1 L total volume):
60 mM Na2HPO4·7H2O 16.1 g Na2HPO4·7H2O
40 mM NaH2PO4·H2O 5.5 g NaH2PO4·H2O
10 mM KCl 10.0 mL 1 M KCl
1 mM MgSO4 1.0 mL 1 M MgSO4
50 mM β-Mercaptoethanol 2.7 mL β-Mercaptoethanol
985 mL Water
.01 % SDS 100 mL 0.1% SDS
6. Add 500 µL cells to 550 µL ZS-buffer.
7. Add 100 µL chloroform to the tubes. Incubate for 2 min at 30°.
8. Add 200 µL 4 mg/mL ONPG to start the reaction. (ONPG should be made fresh each time as 40 mg ONPG in 10 mL sterile water). Note the time of addition precisely. Incubate the reaction at 30° until sufficient yellow color has developed.
9. Stop the reaction by the addition of 500 µL 1 M Na2CO3. Incubate for 5 min at 30°. Note the time of addition precisely. Reaction time is calculated as (stop time)-(start time).
10. Spin out cell debris and chloroform in Table Top centrifuge. Transfer 1 mL of assay mixture to a cuvette.
11. Record the OD420 and OD550 for each tube. The 550 nm reading controls for cell debris and should be small.
11. Calculate units as
(1000)*[ OD420 -1.75* OD550]/[t * v * OD600],
where t is the reaction time in minutes, v is the volume of culture used in mL, and OD600 is the density of the culture.
12. A value of 1.4 OD600 is approximately 0.150 mg protein, and 1 nmol/mL ONPG corresponds to 0.0045 OD420. A total of 1.75 mLs contained the ONPG produced.
13. Convert to Miller Units as (nmol ONPG)/min/(mg protein).