Step 5 instructs you to add 3mL of the diluted thermostable alpha amylase solution.
The recommended way of performing this step (indicated in bold) is by adding the alpha amylase solution immediately as you remove the tube following the 5 minute incubation in the boiling water bath.
We do not recommend allowing time for the solution to cool. When using DMSO to dissolve starch, the product can be very viscous. If the tube is removed from the boiling water bath and allowed to cool (even for 20-30 seconds), the viscous material may not completely dissolve in the added alpha amylase solution.
Therefore, it is important to add the alpha amylase immediately. Vortex immediately after adding the alpha amylase and returning to the boiling water bath for further incubation (6 minutes). Vortex the sample every 2 minutes. These steps are important to ensure the sample remains in solution and does not form a pellet or gel in the tube.
When performing these steps you should observe the sample in the tube.