No DIAPOPS signal above background - No PCR band on the agarose gel
This indicates that the problem resides with the PCR ( 
). The PCR yields no band on the agarose gel either because it is inhibited, because an important reagent is missing from the reaction, or because the reaction condition does not allow the primers to function (missing optimization) ( ). If the PCR does not function at all, it can be checked on the agarose gel. The primer-dimers ( ), which are formed in almost all PCR, will be absent from the agarose gel if inhibition occurs or if elements vital for the reaction, such as the thermostable DNA polymerase, are missing.
The PCR in coated NucleoLink Strips can be inhibited either by residues from the covalent binding of the solid phase primer ( ) or from residues in the sample. The first thing to check is to make a PCR in an uncoated, blocked, NucleoLink Strip with purified DNA ( ). If these reactions yield neither primer-dimers nor PCR bands, an important component was missing or non-functioning, or an inhibitor of the reaction was present in the PCR mix. This can be traced to the following problem:
If PCR in uncoated, blocked, NucleoLink Strips with purified DNA as template shows primer-dimers on the agarose gel ( ), but no specific PCR bands, the PCR is functioning, but the specific amplification is unsuccessful. This may be caused by the following factors:
When the PCR is functioning in uncoated, blocked, NucleoLink Strips with purified DNA as template and the bands on the agarose gel look as expected, the next step is to test the PCR in coated NucleoLink Strips. If the reaction in these strips remains inhibited, which can be seen if the primer-dimers are missing from the analysis on the agarose gel, it is most likely due to residues from the covalent binding reaction of the solid phase primer. This can be caused by on of the following factors:
Link Strips as detected by appropriate bands on agarose gels.