This article is helpful if you've observed a lack of signal in your assay positive controls wells, or you've observed this signal, or Cq value, increase over time.
Never refreeze your dilutions:
Preparing a 1:10 dilution for each assay positive control is required before setting up the plate with qPCR assays. The assay positive control stock tube from Medicinal Genomics consists of a DNA template that represents a small subset of the organisms genome. This DNA is stable in its highly concentrated form, however once it is diluted it becomes much less stable. The added water crystals that occur during the freezing process can sheer and degrade the positive control after diluting. Therefore freezing and thawing this dilution is not recommended.
1 uL is a VERY small volume:
Pipetting 1µL at a time can be very difficult. Increasing the final volume of the dilution from 10µL to 20µL can help. Add 2µL of positive control to 18µL of water could make it work more consistently. Remember to allow assay and control tubes to thaw followed by vortexing and spinning in mini-centrifuge to bring contents to bottom of tube.