Lack of reproducibility in both DIAPOPS and liquid phase PCR can only arise from something which disturbs or inhibits the PCR itself. If an inhibition of the PCR arises from the liquid, it would normally be expected to give a uniform decrease in efficiency in all reactions. An uneven inhibition most likely arises from the single coated NucleoLink wells. This could be due to dust or other contamination of the wells, but also from residues reamaining after the binding procedure of the solid phase primer. The factors in the coating process which could cause problems are:

• Omitting the wash with water after the NaOH wash which follows the coating of solid phase primer
• Omitting the wash after coating of solid phase primer (DP-0058, section 2)
• Coated NucleoLink strips are not blocked with BSA before PCR mix is added

The preparation of the PCR-mix itself could also be the cause of the problem:

• General problems with preparation of PCR mix
• Problems with contamination of old PCR products in preparation of PCR mix
• Using a multipipette 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 forgetting one or two wells.

Finally, it could be the thermal cycling that is not even from well to well. This could be caused by the following factors:

• Omitting to place the silicone spacer plate over the NucleoLink strips in the thermal cycler
• Using a thermal cycler which gives uneven pressure on the NucleoLink Strips

Normally, the problems mentioned with the thermal cycler would also cause evaporation, but not always to an extent where it is clearly visible. It would therefore be beneficial to have a close look at the wells in the NucleoLink Strips after thermal cycling. If no difference in the amount of remaining liquid is observed, evaporation should not be the cause of the reproducibility problems.

Although no evaporation is visible, the strips could still have had an uneven pressure during thermal cycling. In that case the single wells would face different thermal profiles because of different contact areas to the thermal cycler. This could cause the lack in reproducibility.