Talk:Knight:Beta-galactosidase assay/96 well format

This is an outline of various control experiments that I need to do. It is a work in progress and has not been done.

A₆₀₀ versus cell density

1. Grow an overnight culture to saturation in EZ Rich Media
2. Pellet the cells
3. Resuspend in 1/4 of the original volume with EZ Rich Media
4. Add 350 μL of cell suspension to the first well
5. Add 175 μL of previous well to next well.
6. Add 175 μL of EZ Rich Media to that well.
7. Repeat dilution series until the well's solution looks totally clear.
8. Add an addditional well of 175 μL EZ Rich Media.
9. Measure A₆₀₀ of each well in the plate reader.
10. Plot A₄₂₀ versus dilution factor. This relationship should be linear.

β-galactosidase activity versus A₆₀₀ of culture

1. Grow an overnight culture of MG1655 and R2000.E0433
2. In the morning, dilute back both samples into 1mL of EZ Rich Media to an A₆₀₀ of 0.001 (via a Nanodrop reading).
   • Do samples both with and without 1mM IPTG.
3. Prepare many 1.5mL eppendorf tubes with 80 μL of permeabilization buffer.
4. Every hour, measure the A₆₀₀.
5. At each hour point, take three 20 μL samples of culture and add it to 3 tubes.
6. Continue the assay as described at Knight:Beta-galactosidase assay.
7. Plot the β-galactosidase activity in Miller Units as a function of the A₆₀₀ of the culture.

Is there a better way to do this using a plate reader?

A₄₂₀ versus o-nitrophenol concentration

1. Make up a solution of 7 mL to control for background absorbance in β-galactosidase assays
   • 800 μL permeabilization solution
   • 6 mL substrate solution without ONPG
   • 200 μL EZ rich media supplemented with kanamycin and AHL
2. Make up 1mL of 4 mg/mL ONP.
3. Add 350 μL of 4mg/mL solution to the first well.
4. Move 175 μL of previous well to next well.
5. Add 175 μL of background solution to that well.
6. Repeat dilution series until the well's solution looks totally clear.
7. Add an addditional well of 175 μL background solution.
8. Measure A₄₂₀ and A₅₅₀ of each well in the plate reader.
9. Plot A₄₂₀ versus ONP concentration. This relationship should be linear.

Concentration series:

4mg/mL -> 2mg/mL -> 1mg/mL -> 0.5 mg/mL -> 0.25 mg/mL -> 0.125 mg/mL -> 62.5 μg/mL -> 31.25 μg/mL -> 15.625 μg/mL

-> 7.8125 μg/mL -> 3.90625 μg/mL -> 1.953125 μg/mL -> 0.9765625 μg/mL