If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
2. Send the results of the kit standard, blank samples and the results obtained for your sample, in the relevant MegaCalc spreadsheet (if available) to Megazyme ([email protected]). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
3. State the kit lot number being used (this is found on the outside of the kit box).
4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
6. State the sample type and describe the sample preparation steps if applicable.

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

The crucible has the potential to break if heated or cooled too rapidly.  The graph below shows the manufacturer’s recommended heating and cooling profile forsafe use with the crucible. 

Application Note:
Removal of Sucrose, Maltose and Fructose from F3
SUCRASE (MALTASE) + β-GALACTOSIDASE (Lot 111101)
E-SUCRBG
Sucrase (170 U), α-glucosidase (2600 U) and β-galactosidase (3000 U); freeze dried.
For use in the removal of sucrose, maltose and lactose in dietary fibre determinations.  In the Integrated Total Dietary Fibre procedure, in HPLC analysis of non-digestible oligosaccharides using the Waters Sugar-Pak column, the fructosyl-trisaccharide  β-D-Fruf-(2→1)-β-D-Fruf-(2→1)-β-D-Fruf, chromatographs at a similar point to the disaccharides, sucrose, maltose and lactose.  Accurate determination of this trisaccharide requires the hydrolysis of these disaccharides.  This can be achieved using this enzyme mixture.
PROPERTIES
1. ELECTROPHORETIC PURITY:
This is a mixture of sucrase (maltase; from Bacillus stearothermophilis):
- Single major band on SDS-gel electrophoresis (57,750) plus β-galactosidase (from A. niger)
- Single band on isoelectric focusing
2. ACTIVITY:
This enzyme mixture gives complete hydrolysis of sucrose, maltose and lactose under the defined assay conditions, with no hydrolysis of the trisaccharide, β-D-Fruf-(2→1)-β-D-Fruf-(2→1)-β-D-Fruf.
3. STORAGE CONDITIONS:
The enzyme is supplied as a lyophilised powder and should be stored at -20˚C.  On dissolution in buffer or water, the enzyme should be stored in the frozen state.  It is recommended that all buffers used for dilution contain BSA (0.5 mg/mL).
4. PREPARATION OF ENZYME FOR USE:
Dissolve the contents of one vial in 6 mL of 5 mM sodium acetate buffer (pH 5.0).  Transfer aliquots of approx. 2 mL to polypropylene tubes and store at -20˚C between use.  Can be thawed and re-frozen several times.
INCUBATION CONDITIONS:
To 1 mL of sugar mixture obtained in the Integrated Total Dietary Fibre procedure [Step I(b)] containing up to 5 mg/mL of sucrose, maltose and/or lactose and fructotriose,
add:
0.1 mL of sucrase/β-galactosidase enzyme mixture, and incubate at 40˚C, for 60 min.  Terminate the reaction by incubating the tube at 100˚C for 2 min and centrifuge the suspension in a Microfuge at 12,000 rpm for 5 min.
SAMPLE PREPARATION AND HPLC:
Analyse the supernatant solution by HPLC using a Waters Sugar-Pak^® column as described in the Megazyme kit data booklet for the Integrated Total Dietary Fibre method (K-INTDF).  Calculate the amount of fructo-triose by reference to the D-sorbitol internal standard.  This amount should then be added to the determined amount of non-digestible oligosaccharides [NDO; low molecular weight  soluble dietary fibre; Soluble Dietary Fibre soluble in 78% aqueous ethanol (SDFS)].

A volume reduction occurs on mixing water with 95% ethanol (or IMS). 1 L of 78% ethanol is prepared by adding 821 mL 95% ethanol (or IMS) to 207 mL H₂O.
If using a 1 L volumetric flask, this is best accomplished by placing 821 mL 95% ethanol (or IMS) into the volumetric flask. Dilute to volume with deionised water. Mix well. Check the level and if necessary add more deionised water to bring it back up to the 1 L mark.