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.

We feel more comfortable with quadruplicate glucose controls.  If the control is incorrect, or questionable, then all the results are in doubt.

Duplicate samples do not have to be measured.  We just suggest this for laboratories starting up. 

The Regular Maize Starch does not require DMSO pre-treatment.  The value should be about 84% with a moisture content of about 12%, the final dry weight value is about 96-97%.  Store the sample at room temperature, dry.

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 (cs@megazyme.com). 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.

Yes.  We believe that the DMSO step will solubilise vitrified starch in malt.  Make sure that the malt is milled to pass a 0.5 mm screen.  You could vary the time of cooking with DMSO to check solubilisation (i.e. 5 minutes, 10 minutes, or even up to 1 hour).

We think that there is a better chance of success using the Megazyme Starch Damage Kit.

There should be no problem in measuring the starch in plasterboard.  I suggest that you grind about 100 g in a kitchen blender and then fine mill to pass 0.5 mm screen. Run a standard assay, but adjust volume to 10 mL after alpha-amylase treatment. Keep a close check on the pH.  Plasterboard may push the pH value up (pH up to about 8 should be fine).  You may be advised to run a DMSO format concurrently just to be sure.  When you treat with amyloglucosidase, I would advise that you take 0.2 and 0.4 mL aliquots of digest (to get the colour up), also, be careful about checking the pH.

A 20% fat content could cause a problem for the method.  We suggest that the sample be defatted before analysis for starch.

Our Total Starch Assay Kit could measure starch left in a residue, or starch extracted. No method could measure potential extractable starch, as this will depend on numerous factors, including processing equipment, conditions etc.

You can use phosphate buffer at the same concentration.

Most of the maltodextrins can be removed with 50% ethanol washing.  If the starch is not gelatinised, it can be washed with cold water.  This will remove all of the soluble maltodextrins, but the starch will spin down.  If the starch has been gelatinised, then the best material which can be used for washing is 50% ethanol.

Yes, the Total Starch Kit can be used to measure starch in roots and shoots etc. 

We think that calcium carbonate etc. will not cause any problems.  However, this of course depends on the amount present and if it changes the pH of the incubation mixture.

The method will work for some chemically modified starches (e.g. crosslinked) however, if the degree of chemical modification is high, there will be an underestimation as the modification will interfere with complete hydrolysis to glucose and subsequent measurement.

You only need to wash samples which you feel may contain glucose and/or maltodextrins, e.g. breakfast cereals.  There is little glucose in ground cereals, so it is not necessary to pre-wash these materials.

The enzymes from this kit are stable at room temperature for at least 6 months.  At 4˚C, they are stable for several years.

The Starch Damage Kit may be best for this.  If the starch is gelatinised and dried before analysis the correct results for gelatinisation will not be obtained.

The Total Starch Kit can accurately measure starch levels as low as 1% w/w.

The absorbance for 100 micrograms of glucose (in 3 mL of GOPOD Reagent) is about 0.97.

Yes, you can reduce the volume of GOPOD to 1 mL and use micro cuvettes.  This will increase sensitivity by ~ 3-fold.

DMSO does solubilise resistant starch (crystallise amylose and amylopectin).  The only starch material we have had problems in dissolving in DMSO is potato amylose.

We believe that for starch fragments, oligosaccharides of a DP up to 10 would be soluble in 80% alcohol.  The degree of solubility of other oligosaccharides would depend on the sugar type and linkage type.

The Total Starch Kit can be used for liquids containing as little as 200 micrograms per mL with some adjustments of conditions, as below:
Mix 0.5 mL of sample with 0.5 mL of 100 mM sodium acetate buffer (pH 4.5). Incubate at 40˚C and add 0.1 mL of Amyloglucosidase and incubate for 30 minutes.  Add GOPOD reagent as usual.  You will need to run an AMG blank as this enzyme preparation contains a very small amount of glucose.

The AMG activity was determined with soluble starch as substrate (10 mg/mL) in 0.1 M sodium acetate buffer at pH 4.5 and 40˚C.  One Unit is the amount of enzyme required to hydrolyse one micromole of maltose per minute (i.e. to release 2 micromoles of glucose).  Glucose release is measured with Glucose Determination Reagent.

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