In the trouble shooting of this problem, the background is assumed to be derived from the detection step after PCR, and not from the PCR reaction itself, or from the coating of the solid phase primer. However, in the Nunc A/S research laboratory examples have been observed, where high background signals were arising from the coating:

• The solid phase primer recognizes streptavidin-AP and yields high background signals

However, the most likely cause of high background is if a problem occurs in the detection step after PCR. The following factors can give rise to high background signals in this step:

• Concentration and temperature of the washing buffer used after hybridization are not optimal for the detection probe
• In the wash after hybridization normal Tris wash buffer is used
• Incorrect incubation buffer (e.g. PBS or BSA in buffer) when adding streptavidin-enzyme conjugate
• Incorrect incubation conditions during substrate reaction
• The substrate reaction is not terminated before measuring. Conversion of substrate may continue during reading and yield different results from one end of the MicroWell^® plate to the other as well as between plates.
• Improper settings of blank values in the plate reader
• In the various washing steps the washer does not wash all wells properly
• In general, the blocking and washing volumes are inadequate in relation to incubation volumes
• Problems in the general washing procedure