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 &mu;L of cell suspension to the first well
 * 5) Add 175 &mu;L of previous well to next well.
 * 6) Add 175 &mu;L of EZ Rich Media to that well.
 * 7) Repeat dilution series until the well's solution looks totally clear.
 * 8) Add an additional well of 175 &mu;L EZ Rich Media.
 * 9) Measure A600 of each well in the plate reader.
 * 10) Plot A420 versus dilution factor. This relationship should be linear.

Effect of evaporation on absorbance readings




Effect of evaporation on A600 readings

 * 1) Aliquot 50 &mu;L of culture into an entire row of wells.
 * 2) *Do duplicates rows to assess measurement variability in duplicate samples.
 * 3) Aliquot 100 &mu;L of a of culture into a second row of wells.
 * 4) Add increasing volumes of water to each well (by 10 &mu;L increments).
 * 5) Measure the A600 of the plate.

Effect of evaporation on A420 readings

 * 1) Aliquot 50 &mu;L of a set concentration of ONP into an entire row of wells.
 * 2) *Could add different concentrations of ONP to different rows.
 * 3) Add increasing volumes of water to each well (by 10 &mu;L increments).
 * 4) Measure the A420 of the plate.

&beta;-galactosidase activity versus number of cells

 * 1) Do parallel &beta;-galactosidase assays with a variable volumes of cells from the same grown culture.
 * 2) *1 = 1 &mu;L of cells
 * 3) *2 = 2 &mu;L of cells
 * 4) *3 = 3 &mu;L of cells
 * 5) *4 = 4 &mu;L of cells
 * 6) *5 = 5 &mu;L of cells
 * 7) *6 = 5 &mu;L of media

&beta;-galactosidase activity versus growth phase of culture

 * 1) Grow an overnight culture of several constructs
 * 2) *A = P20060 in MG1655
 * 3) *B = P20060 in MG1655+IPTG
 * 4) *C = P20060+IPTG+AHL
 * 5) *D = P20060.E0433+IPTG
 * 6) *E = P20060.E0433+IPTG+AHL
 * 7) *F = R2000.E0433+IPTG+AHL
 * 8) *G =
 * 9) *H = Media+IPTG+AHL
 * 10) In the morning, dilute back samples into EZ Rich Media to an A600 of 0.001 (via a Nanodrop reading) in tubes (5 mL of culture in a 14mL tubes per construct).
 * 11) Let grow 1 hour.
 * 12) Add IPTG and/or AHL to cultures as appropriate.
 * 13) Prepare 8mL of permeabilization buffer.
 * 14) Every hour
 * 15) Take 175 &mu;L of each culture and put it in the appropriate row and column of a 96 well plate (A600 plate).
 * 16) Measure the absorbance at 600nm in the plate reader of the newest column of the A600 plate.
 * 17) Aliquot 80 &mu;L permeabilization buffer into each well of the corresponding column of a second 96 well plate (permeabilization plate).
 * 18) Take 20 &mu;L of culture from the most recent column of the A600 plate and add it to the corresponding column of the permeabilization plate.
 * 19) Parafilm that column of the permeabilization plate.
 * 20) Once each time point has been taken, move 25 &mu;L of each well from the permeabilized culture to a new plate.
 * 21) Add 150 &mu;L of substrate solution to each well.
 * 22) Place plate in the plate reader to measure change in A430 as a function of time.
 * 23) Plot the &beta;-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 14 mL to control for background absorbance in &beta;-galactosidase assays
 * 2) *1600 &mu;L permeabilization solution
 * 3) *12 mL substrate solution without ONPG
 * 4) *400 &mu;L EZ rich media supplemented with kanamycin and AHL
 * 5) Make up 1mL of 1 mg/mL ONP.
 * 6) Add 350 &mu;L of 1mg/mL solution to the first well.
 * 7) Move 175 &mu;L of previous well to next well.
 * 8) Add 175 &mu;L of background solution to that well.
 * 9) Repeat dilution series until the well's solution looks totally clear.
 * 10) Add an additional well of 175 &mu;L background solution.
 * 11) Measure A420 of each well in the plate reader.
 * 12) Plot A420 versus ONP concentration. This relationship should be linear.


 * Reshma 13:16, 13 November 2007 (CST): I seem to get different calibration curves every time I do this experiment presumably due to errors in measurement of the ONP (since I am measuring small amounts).