GFP-sacB Characterization

Inoculate cells with GFP-sacB gene from the cell stock.

Add 5mL of LB media and 5µL of the appropriate antibiotic to a Falcon tube.

Incubate the Falcon tube at 37°C in an incubator.

Decide the concentrations of IPTG and sucrose solutions for characterization.

Prepare new Falcon tube(s).

Add 5mL of M9 media, 5µL of the appropriate antibiotic, and 100µL of cells cultured the previous day.

Add the required volume of IPTG to reach the desired concentration.

Incubate the cells for 2h 0m 0s at 37°C.

Prepare a sterile 96-well plate.

Design an appropriate plate map, each sample (with a particular IPTG and sucrose concentration) must be repeated 3 times. Example of plate map:

Adjust sucrose concentration by adding DI water and sucrose solution to each well (final volume of 20µL) according to the plate map.

Add 200µL of cultured cells to each well already containing DI water and sucrose solution (final well volume of 220µL).

Add at least 3 wells of 220µL of M9 media as the blank (negative control).

Place the 96-well plate into the plate reader.

Create a protocol in the plate reader's software according to the following setting:

A	B
Plate Type	96 WELL PLATE
Set Temperature	Setpoint 37°C
	Preheat before moving to next step
Start Kinetic	Runtime 6:10:00 (HH:MM:SS), Interval 0:30:00, 13 Reads
Shake	Orbital: Continuous
	Frequency: 282 cpm (3 mm)
Read 1	Absorbance Endpoint
	Full Plate
	Wavelengths: 600
	Read Speed: Normal, Delay: 100 msec, Measurements/Data Point: 8
Read 2	Fluorescence Endpoint
	Full Plate
	Excitation: 485, Emission: 528
	Optics: Top, Gain: 100
	Light Source: Xenon Flash, Lamp Energy: High
	Read Speed: Normal, Delay: 100 msec, Measurements/Data Point: 10
	Read Height: 7 mm
End Kinetic

Start the continuous reading.

After the reading is complete, remove the 96-well plate from the plate reader.

Save the data as a CSV file for future analysis.

Discard the used 96-well plate in a biohazard bin.