Functional Assay for PCB
This protocol was developed from the Quail Lab at UC-Berkely.
ONPG assay from Yeast Protocols Handbook from Clontech Laboratories
1. Inoculate 5 mL culture of L6457 yeast strain with BNT and GP3 in SD (leu- trp-). Leave overnight until saturated (dense culture, about OD600≈2)
2. In a 48-well plate, grow pre-cultures in 4 wells (4 wells for culture and 4 wells for experiment). In each well have the following set-up [Want PCB conc. to be 2 μL/mL]:
- a. 374 μL SD (leu- trp-); 1 μL PCB; 125 μL starter culture
- b. 375 μL SD (leu- trp-); 125 μL starter culture
3. Let this grow for 16-24 hours in a shaker either in darkness or far-red light
4. Do a 1:4 dilution into YEPD culture, 2 different wells as shown below:
5.Grow up for 5 hours, in their respective conditions in the shaker
6. While growing for 5 hours put ONPG into Z-Buffer (4 mg/mL), vortex for 1 minute
7. Take the OD600 of each culture. Should be between 0.5-0.8. (Remember to blank with YEPD w/ PCB for cultures that have PCB in it as PCB absorbs at 600nm).
8. Pool the 4 of each type into a 1.5 mL eppendorf tube. Note the total amount of pooled cells.
9. Centrifuge for 30 seconds at 14,000 rpm (10,000 x g). Discard supernatant.
10. Resuspend with 1.5 mL of Z-Buffer.
11. Centrifuge for 30 seconds at 14,000 rpm (10,000 x g). Discard supernatant.
12. Resuspend each pellet in 300 μL of Z-Buffer. (Note that this is a 5-fold concentration, 1.5 mL to .3 mL).
13. Transfer 0.1 mL of the cell suspension to a fresh eppendorf tube.
14. Place cells in liquid nitrogen until the cells are frozen (0.5-1 min).
15. Place frozen tubes in a 37°C water bath for 0.5-1 min to thaw.
16. Repeat the freeze/thaw cycle (Steps 14 and 15) two more times to ensure that the cells have been lysed.
17. Set up a blank tube with 100 μL of Z-Buffer.
18. Add 0.7 mL of Z-Buffer + β-mercaptoethanol to the reaction and blank tubes.
19. Start timer, and immediately add 160 μL of ONPG in Z-Buffer to the reaction and blank tubes.
20. Place tubes in a 30°C incubator.
21. After the yellow color develops, add 0.4 mL of 1 M Na2CO3 to the reaction and blank tubes. Record elapsed time in minutes.
- a. The time needed will vary, no longer than 24 hours for a very weak reaction.
- b. The yellow color is not stable and will become more intense with time.
22. Centrifuge tubes for 10 minutes at 14,000 rpm to pellet cell debris.
23. Using a spectrophotometer, measure the OD420 of the samples.
24. Calculate the β-galactosidase Units
- a. 1 unit of β-galactosidase is defined as the amount which hydrolyzes 1 μmol of ONPG to o-nitrophenol and D-galactose per min per cell (Miller 1972; Miller 1992):
Where:
V = 0.1 mL * Concentration Factor ^
OD600 = A600 of 1 mL of culture^ The concentration factor from step 12 usually calls for a conc. factor of 5, but in case this is not the case, please take this into account. Also if need to dilute cell culture to remain within the linear range, it should be taken into account here as well.
