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.

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.

Amylose is always reported as a percentage of the starch, not as a percentage of the sample weight. The method should be followed as described.

We do not recommend K-AMYL for starch measurement. The aim with this method is to get a representative sample of the starch to get the amylose content. For accurate starch determination, we recommend using K-TSTA.

We think that the only effective method to separate amylose and amylopectin is by gel filtration.

Mill with Retsch 0.5 mm screen.

The method should be fine for amylose/amylopectin in potato starch.

Reproducibility +/- 2 to 4% of the value.

Yes, there should be no problem in distinguishing samples with these differences in amylose content.

Solution not always clear A5 – this is ok.  Solution not always clear A7 – this is ok as well.

The starch control is high amylose maize starch.

We suggest that you make the following changes:
1. A2 – use 2 mL DMSO
2. A4 – use total of 12 mL Ethanol (4 + 8– need longer tubes)
3. A6 – use 2 mL of DMSO
4. A7 – use 4 mL of Con A solvent.
Of these, the changes in A6 and A7 are probably the most important. 

The Amylose/Amylopectin kit employs Con A to selectively precipitate the amylopectin.

Speed of centrifugation is the most important parameter.  We use the microfuge at room temperature.

The white precipitate is most probably protein, and this will cause no real problems. However, an alternative is as follows:
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Step 2: Dissolve in 2 mL DMSO. Centrifuge at high speed (could use a micro-centrifuge – 14,000 rpm, but bench centrifuge – 3,000 rpm, may be OK) and recover the supernatant.  Treat this as follows:
Step 4: Add 4 mL ethanol, then 8 mL of ethanol.
Step 6: Use 2 mL of DMSO.
Step 7: Use 4 mL of Con A solvent and then make up to 25 mL as per method.

Rice starch should dissolve completely, but the solution may not be "crystal clear".  In fact, it generally isn’t.  With rice flour, the solution will be quite turbid due to materials other than starch, which will not dissolve.  You can also increase the amount of DMSO used in the test.  We have now introduced this modification into the method.