Lack of reproducibility in the DIAPOPS results but not on the agarose gel analysis of the liquid phase PCR products indicates that the PCR is working as expected, but that the problem is in the detection of the solid phase product by hybridization after PCR.

Problems associated with binding of the solid phase primer before PCR would result in a uniform change of the DIAPOPS results. Insufficient washing after the binding procedure with the solid phase primer would be reflected by inhibition of the PCR which would be detected by the agarose gel analysis of the liquid phase PCR product.

The most likely reason for the lack of reproducibility in the DIAPOPS results is in the hybridization detection procedure of the solid phase product after the PCR. The following factors may cause the problem:

• 25 µl of 40 mM EDC is added twice in the covalent coupling procedure of the solid phase primer
• In the washing step after PCR incorrect concentrations of reagents are used
• In the washing step after PCR, the Immuno-washer is not flushed with TRIS wash buffer after the NaOH wash
• In the wash after hybridization normal Tris wash buffer is used
• The substrate reaction is not stopped 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.
• Before reading, it has not been checked if salt from e.g. NaOH is dried out on the outside of the bottom of some of the NucleoLink wells. This may prevent measuring of light absorption through the well.
• 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
• Using a multi-channel pipette where one pipette tip is not adding the correct amount of liquid.
• Using a single pipette for all wells in a plate, causing different amounts of liquid to be added to the NucleoLink wells, or omitting one or two wells.