To prepare Solution 1: Acetate Buffer, dissolve the contents of Bottle 1 (20mL) to 400mL in distilled water.

Store at 4°C

To prepare Solution 2: Beta-fructosidase, dissolve 1mL of solution from Bottle 2 (20mL) to 10mL in distilled water.

For convenience, prepare ten 1mL aliquots of this. Store them at -20°C

To prepare Solution 3: GOPOD Reagent Buffer, dissolve 5mL of solution from Bottle 3 (50mL) to 100mL in distilled water.

Divide Solution 3 into two portions of 20mL and 80mL .

Solution 3 must be used immediately and can't be stored.

To prepare Solution 4: GOPOD Reagent, dissolve all of the contents of Bottle 4 into the 20mL portion of Solution 3.

For convenience, divide this into twelve aliquots of 1.65mL each as working stocks.

Cover completely in aluminium foil and store at -20°C .

Add 1.632mL from the 1.65mL aliquots of the working stock to the 80mL portion of Solution 3. This is your Solution 4 (GOPOD Reagent).

Cover completely in aluminum foil and store at 4°C .

To prepare fresh Solution 4 (GOPOD Reagent) in the future from the working stock aliquots:

First prepare 80mL of fresh Solution 3 (GOPOD Reagent Buffer) by dissolving 4mL of the solution from Bottle 4 to 80mL in distilled water.

Add 1.632mL from the 1.65mL aliquots of the working stock to this 80mL of freshly prepared Solution 3

Verify that the absorbance of Solution 4 at 510 nm against a distilled water blank is <0.05,

Note: Solution 3 cannot be stored and must be prepared freshly to make a fresh batch of Solution 4 (GOPOD Reagent).

100ug D-Glucose standard:

Take 0.1mL of the solution from Bottle 5 in a test tube.

Add 0.3mL of distilled water.

Sample:

Dilute your sample according to the directions in the kit instructions or by any factor that would bring your expected sucrose+glucose concentration to the range of 0.05 g/L to 1 g/L (as per our standardization curve).

Take 0.1mL of the diluted sample in a 1mL MCT, and add 0.1mL of Solution 1 (Acetate buffer). Denote as Solution A.

Take 0.1mL of the diluted sample in another 1mL MCT, and add 0.1mL of Solution 2 (Beta-fructosidase). Denote as Solution B.

Blanks:

In a 1mL microcentrifuge tube (MCT), add 0.1mL of distilled water and add 0.1mL of Solution 1 (Acetate buffer). This is the blank for Solution A.

In another 1mL MCT, add 0.1mL of distilled water and add 0.1mL of Solution 2 (Beta-fructosidase). This is the blank for Solution B.

Incubate the 100 ug D-glucose standard, blanks, and samples in a water bath at 50°C for 0h 20m 0s.

Add 1.5mL of Solution 4 (GOPOD Reagent) to the 100 ug D-glucose standard, blanks, and sample A and B solutions.

Incubate in a water bath at 50°C for 0h 20m 0s.

Solutions that contain free D-glucose or sucrose that was inverted into D-glucose by the action of beta-fructosidase should turn red.

Pipette 0.3mL from each of the reaction mixtures (standard, blanks, sample A and B solutions) into the wells of a 96 well plate.

Measure the absorbance of the reaction mixtures at 510 nm in a 96 well plate reader.

Otherwise, a regular cuvette and spectrophotometer can be used to measure absorbance.

Define ∆A as the absorbance of Solution A of the sample against the acetate buffer blank and ∆B as the absorbance of Solution B of the sample against the beta-fructosidase blank.

Define factor to convert from absorbance to ug for 100ug of D-glucose as F.

F = 100/absorbance of the 100ug D-glucose standard.

Define D as the factory by which the sample was diluted. For example, if 1 mL of the sample was diluted to 100mL with distilled water, D = 100.

Sucrose concentration is given by:

(∆B-∆A) x F x D x 0.0095

See the Megazyme kit instructions for the derivation of the above formula.