In the trouble shooting of this problem it is assumed that the liquid phase PCR is normal and the DIAPOPS results are normal in the first detection.

This effect is the expected result of rehybridizations, as the solid phase DNA slowly deteriorates in the multiple handling steps ( ). However, the solid phase DNA is very robust when stored without liquid ( ). The only measurable decrease in signal in a subsequent hybridization is seen when the NucleoLink Strips are stored with liquid between hybridizations ( ). The following factor is the main reason for the decrease in DIAPOPS signal in a subsequent hybridization:

• After hybridization, the NucleoLink strips are stored with liquid

Other factors are also important, but will not give a decrease in signal by themselves, but rather a reproducibility problem. These factors are:

• Problems with storage and handling of primers and probes (e.g. too many freeze thaw cycles of the same stock solution)
• The incubation time and temperature in the incubation with streptavidin-enzyme conjugate are inadequate