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 several constructs
   • A = MG1655
   • B = MG1655+IPTG
   • C = P20060+IPTG+AHL
   • D = P20060.E0433+IPTG
   • E = P20060.E0433+IPTG+AHL
   • F = R2000.E0433+IPTG+AHL
   • G =
   • H = Media+IPTG+AHL
2. In the morning, dilute back samples into EZ Rich Media to an A₆₀₀ of 0.001 (via a Nanodrop reading) in a 96 well plate. (Each well in a row should be identical with 175 μL of culture according to the list above.)
3. Let grow 1 hour.
4. Add IPTG and/or AHL to rows as appropriate.
5. Prepare the permeabilization buffer and aliquot 80 &muL into each well of a 96 well plate.
   • Using a larger volume for permeabilization step because the samples are sitting for a while.
6. Measure the A₆₀₀ of the plate every 30-60 minutes or grow the plate in the plate reader and pause it every 30-60 mins.
7. At each hour point, take 20 μL of the next column of culture and add it to the corresponding columns of the permeabilization plate.
8. Once each time point has been taken, move 25 μL of each well from the permeabilized culture to a new plate.
9. Add 150 μL of substrate solution to each well.
10. Place plate in the plate reader to measure change in A₄₂₀ as a function of time.
11. Plot the β-galactosidase activity in Miller Units as a function of the A₆₀₀ of the culture.

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