IGEM:IMPERIAL/2009/M0/Assays/Glucosesensing

=Glucose Assay=

Aims

 * To detect the changes in the concentrations of glucose over time
 * Range is from twice as high as normal LB media to glucose levels after overnight culturing

Assay
Glucose Enzyme Mix specifically oxidizes glucose to generate a product which reacts with a dye to generate color (λ = 570 nm) and fluorescence (Ex/Em = 535/587 nm). The generated color and fluorescence is proportionally to the glucose amount. The kit detects 1‐10000μM glucose samples.

Equipment

 * Fluorimetric Plate reader

Reagents
Kit Contents Glucose Assay Buffer		25 ml Glucose Probe (lyophilized)		1 vial Dimethylsulfoxide (DMSO; Dried)	0.4 ml Glucose Enzyme Mix (lyophilized)	1 vial Glucose Standard (100 nmol/μl)	100 μl

Reagent preparation
protect from light and moisture. Use within two months. 20°C. Keep on ice while in use. Use within two months.
 * Glucose Probe: Dissolve in 220 μl DMSO (provided) before use. Store at –20°C,
 * Glucose Enzyme Mix: Dissolve in 220 μl Glucose Assay Buffer. Aliquot and store at –

Standard curve preparation
1.	dilute the Glucose Standard solution to 0.1 nmol/μl by adding 10 μl of the Glucose Standard to 990 μl of Glucose Assay Buffer, mix well.

2.	Take 20 μl of diluted glucose standard and add into 180 μl of Glucose Assay Buffer. Mix well.

3.	Add 0, 2, 4, 6, 8, 10 μl into each well individually. Adjust volume to 50 μl/well with Glucose Assay Buffer to generate 0, 0.2, 0.4, 0.6, 0.8, 1.0 nmol/well of the Glucose Standard.

Sample preparations
Prepare test samples in 50 μl/well with Glucose Assay Buffer in a 96‐well plate

Fluorimetric Assay
1.	For each well, prepare a total 50 μl Reaction Mix containing: 46 μl Glucose Assay Buffer 2 μl Glucose Probe 2 μl Glucose Enzyme Mix Mix well Note: Use 0.4 μl of the probe per reaction will decrease the background reading significantly to increase the detection sensitivity 2.	Add 50 μl of the Reaction Mix to each well containing the Glucose Standard and test samples. Mix well

3.	Incubate the reaction for 30 minutes at 37°C, protect from light.

4.	Measure Ex/Em = 535/590 nm for fluorometric assay in a microplate reader.

Calculations: Correct background by subtracting the value derived from the 0 glucose control from all readings (Note: The background reading can be significant and must be subtracted from sample readings).

Glucose concentrations of the test samples can then be calculated.

C = Sa/Sv (nmol/μl or μmol/ml, or mM)

Where Sa is sample amount (in nmol) from standard curve. Sv is sample volume (in μl) added into the sample wells. Glucose Molecular Weight 180.16. Example of glucose standard curve: