In the trouble shooting of this problem, it is assumed that the liquid phase PCR is normal and that the DIAPOPS results are normal or perhaps a bit lower than normal in the first detection.

At the Nunc A/S research laboratory rehybridizations has often been examined. Contrary to expectation it has occasionally been seen that DIAPOPS signals increase in subsequent hybridization's ( ). The most likely cause is that the first wash after PCR is inadequate ( ). An insufficiency in this step would cause a lower signal in the first detection as not all solid phase products were single stranded and ready for detection by the hybridization probe. In the denaturation after hybridization, the hybridized probe and the remaining double stranded solid phase products will be denatured and washed off. For this reason, more solid phase products are available for detection in the second hybridization ( ). The following factors can be responsible for the problem:

• In the washing step after PCR incorrect concentrations of NaOH or Tween 20 were used
• Insufficient washing after PCR did not denature all solid phase strands, and the solid phase strands may not all have been detected by the hybridization probe
• In the washing step after PCR, the strips were not washed with NaOH, e.g. using Tris wash buffer instead of NaOH
• In the various washing steps the washer did not wash all wells properly
• Problems in the general washing procedure

Furthermore, the temperature control in the incubation step with streptavidin-enzyme conjugate can influence the intensity of the DIAPOPS signal.

• The incubation time and temperature in the incubation with streptavidin-enzyme conjugate are inadequate