1. Introduction
2. Blocking with Tween 20
3. Blocking with BSA
   1. DIAPOPS
   2. Liquid phase PCR products
4. Difference in BSA blocking conditions
5. Conclusion

1. Introduction
When the NucleoLink Strips have been coated with the solid phase primer ( ), they can be stored for a prolonged time ( ). However, before they are ready for PCR, a pre-PCR blocking is necessary. The necessity of this blocking is not dependent of the time on the storage, as even newly coated Strips must be blocked. It is not possible to block the Strips before storage ( ).

2. Blocking with Tween 20
After the covalent binding with the solid phase primer, the NucleoLink Strips are washed ( ) with 0.4 N NaOH containing 0.1 % Tween 20 ( ), followed by a wash with distilled water ( ). The final wash with distilled water removes salt residues and ensures that the surface is as clean as possible before storage. The detergent in the NaOH acts as a blocking component, to ensure that no adsorption to the wall occurs after the covalent binding of the solid phase primer. However, inconsistent results were observed with NucleoLink Strips which were only blocked with Tween 20, particularly in the detection of samples with low concentration of template DNA. One reason for this could be that the detergent blocking layer is insufficient during the thermal cycling because the NucleoLink Strips are exposed to high temperatures. Another reason could be that the last step in the coating procedure, i.e. the water wash, removes too much of the detergent, thus diminishing the blocking effect.

3. Blocking with BSA
The effect of BSA blocking before addition of the PCR mix to the coated NucleoLink Strips was examined in both the DIAPOPS results ( ) and in the liquid phase of the DNA amplification, illustrated with agarose gels ( ).

3 a) DIAPOPS
The blocking effect of Tween 20, taking place in the washing step after coating, was not sufficient. Therefore, blocking with BSA was tested. Concentrations of 2 and 10 mg/ml were used. The blocking was carried out for either 30 or 60 minutes, with or without agitation during blocking. All blockings were performed at room temperature. After blocking with BSA, the DIAPOPS results were greatly improved; both the detection limit and the dynamic range of the results were enhanced. Figure 1 shows a typical response of a pre-PCR blocking with 10 mg/ml BSA incubating with agitation for one hour. It has also been observed that the same positive results can be acquired by adding 1 mg/ml BSA to the PCR mix ( ), although this approach has not been thoroughly tested yet.

Figure 1: DIAPOPS results as a function of template dilution. DIAPOPS was carried out on NucleoLink Strips with and without pre-blocking with BSA before PCR. The positive effect of the BSA blocking clearly yields an increased signal level and enhanced limit of detection.

3 b) Liquid phase PCR products
The effect of the BSA blocking on the concentration of the liquid phase PCR products is illustrated in Figure 2. When there is no BSA blocking before PCR, it is not clear that is was a dilution series that was initially added as templates. The three first reactions yields high uniform product concentrations, and the lower template concentrations yield almost identical product concentration, even though there is a large difference in the template number. When the NucleoLink Strips has been pre-blocked with BSA, the correlation between PCR product concentration and template concentration is more obvious, and the dilution series is yielding very good results on the agarose gel.

Figure 2: Shows an agarose gel analysis of the liquid phase PCR product from a DIAPOPS analysis of a dilution series of BLV plasmid DNA ( ) . In the gel on the left is seen PCR products from a NucleoLink Strip not blocked with BSA before addition of PCR mix, and in the gel on the right is seen PCR products from a NucleoLink Strip blocked with BSA as described in the DIAPOPS procedure ( ) . In this experiment, contamination with old PCR products can be seen in the blank samples ( ) .

4. Difference in BSA blocking conditions
No significant difference in DIAPOPS results was seen between the different conditions, regarding time and concentration of BSA, used in the blocking with BSA, but to ensure that the blocking is sufficient, the recommended method is to add 200 µl of TRIS wash buffer with 10 mg/ml BSA and incubate with agitation for one hour ( ).

5. Conclusion
It is necessary to block the coated NucleoLink Strips with BSA before running the PCR. The best method is to add 200 µl DIAPOPS wash buffer with 10 mg/ml BSA and incubate with agitation for 1 hour at RT.

The pre-PCR BSA blocking greatly improves the DIAPOPS results and improves the correlation between the liquid phase PCR product concentration and the amount of added template DNA.