General handling of NucleoLink Strips in DIAPOPS

) the NucleoLink Strips are used as a solid support for solid phase DNA amplification (

). The strips are employed as the only vessel necessary in the procedure from coating to detection. There are several important issues in the practical handling of NucleoLink Strips which are important in order to achieve good results, which are basically identical to the normal manipulation of MicroWell^® Plates.

2. The term: Wash three times

This term means that the wells must be filled with washing liquid or washing buffer and emptied. This step should be repeated three times. After the last wash, the wells should be completely empty (

) before they are ready for addition of a new incubation buffer. All washing steps in the DIAPOPS procedure are composed of three washes, followed by a soak for 5 minutes and ending with three additional washes. It is important to follow this washing procedure carefully, to avoid high background signals. Inadequate washing can influence the background severely due to the high sensitivity of the DIAPOPS analysis (

3. Volume of washing liquid
It is recommended that the volume of the washing buffer should exceed the volume of the incubation liquid. A volume of 100 µl is added to each well in the covalent binding of the solid phase primer at the beginning of the DIAPOPS procedure (

). The volume of the washing liquid after coating, as well as in all other washes, should be more than 100 µl to ensure that all the reagent is flushed away. Therefore, a volume of at least 150 µl or 200 µl is suggested in the washing procedure. This will ensure that any liquid on the sides of the wells is washed off.

4. Tools for washing
When using a normal multichannel pipette (

) or even a single pipette to wash the strips, the wells should be closely scrutinized to ensure that they are all washed, each time. It is recommended that special wash equipment should be used when handling more than a few plates. The Nunc-Immuno Wash (

) (Nunc-Immuno Wash 8: Cat. No. 470173. Nunc-Immuno Wash 12: Cat. No. 455792), or an automated MicroWell^® plate wash station can be used. The use of an ImmunoWash or an automated wash station has the advantage, in addition the ability to wash many plates rapidly, that the walls of the wells are flushed with liquid when the wells are filled with washing solution. Moreover, it is more likely that the wells are totally filled during washing ensuring that the amount of washing liquid exceeds the volume of the incubation buffer.

5. Emptying the NucleoLink Strips
When washing the NucleoLink strips with a Nunc-Immuno Wash, the strips are emptied by suction between each wash. However, when the wells are washed, they are not completely empty as neither a pipette nor a wash can achieve this. Therefore, it is necessary to completely empty the wells after the washing by hitting them against a folded towel several times with the opening facing the towel. During this procedure the last remaining liquid is efficiently removed. In order to assure that the wells are as empty as possible the hitting should be rather vigorous. Since the Strips are locked very securely in the frame, this hitting should not dislodge the Strips from the frame.

6. The NucleoLink frames
The red NucleoLink frame locks on to every well in each NucleoLink Strip (

). The NucleoLink Strips can fit into the frame in only one direction. It is possible to write on the small, flat square on the end NucleoLink Strips (

) as well as on the red frame. If a NucleoLink Strip should accidentally break, the single wells will still fit into the frame, and can be handled. The red NucleoLink frames are not heat-stable, and will melt if they are inserted into the thermal cycler. The frame with the NucleoLink Strips must therefore be removed before insertion of the Strips into the thermal cycler.

7. Sealing of NucleoLink Strips during DIAPOPS and storage
During thermal cycling the NucleoLink Strips are sealed with the heat-stable Tape 8 (Nunc Tape 8, Cat. No. 249719) to avoid evaporation. It is very important to assure that all wells are total closed when sealing the NucleoLink Strips with Tape 8. Tape 8 is designed to cover only one Strip. This is important, as the Strips are separately inserted to the thermal cycler (

), and a broader tape would create problems by making the Strips adhere to each other. However, this small design of the tape makes it very important to assure that all wells are totally sealed.

As an alternative, it is possible to use the GeNunc Tape 48 (Nunc GeNunc Tape 48, Cat. No. 232700), which is also heat-resistant. However, use of the GeNunc Tape 48 demands some practice in inserting and removing the Strips from the thermal cycler. The Nunc Sealing Tape (Cat. No. 236366), which is the other tape used in the DIAPOPS procedure (

), is not heat-stable, and cannot be used in a thermal cycler. However, this tape is very practical for sealing the Strips during prolonged incubation at 50ºC which is used during covalent coupling of the solid phase primer (

) and during hybridization (

7 a). Evaporation during incubation
It is necessary to seal the NucleoLink Strips with tape. If the Strips are only closed with a lid, up to 10% evaporation will occur even for a short incubation period of one hour, as indicated in Figure 1. Closure with a lid (Nunc Lid for MicroWell^® Plates, Cat. No. 263339) can only be used for a very short incubation e.g. 30 minutes as used during substrate incubation (

Figure 1: Illustrating the degree of evaporation in percent from NucleoLink Strips incubated at 50ºC, sealed either with Nunc Sealing Tape or only with a lid. A volume of 50 µl was initially added to the wells, and the evaporation was measured as the weight loss after incubation. The incubation was carried out in a incubator with a ventilator to ensure a uniform temperature. This explains why the evaporation is so high. It can be concluded that it is necessary to seal the Strips with tape, even if the incubation is only for one hour. The final measurement at 5 hours was made on Strips, which were not placed at the same site in the incubator as the other Strips, which explains the decrease in the rate of evaporation at this time. Evaporation is very dependent upon the airflow, which is not identical in different parts of the heating incubator.

7 b). Sealing during storage
The NucleoLink strips can be stored at 4ºC after coating (

). Furthermore, after the first detection of the solid phase PCR product (

), the Strips can be stored for further analysis using rehybridization (

). During storage the strips must be totally empty. Otherwise, the DNA will degrade (

). It is not necessary to dry the NucleoLink Strips with e.g. heat. A thorough emptying as previously described (

) is sufficient. Sealing the NucleoLink Strips with tape during storage is not recommended, as condensation may occur inside the wells. No difference has been observed between Strips stored in sealed bags, and Strips stored in bags which were only folded. At Nunc A/S Research Laboratory, the NucleoLink Strips with covalently bound DNA are normally stored at 4ºC in unsealed polyethylene bags.

8. Removal of tape from NucleoLink Strips
To avoid tearing the NucleoLink Strips from the frame, when the tape is removed from the Strips after thermal cycling (

), care must be taken not to remove the tape too rapidly. A finger may be used to secure the Strip in the frame while removing the tape (

). The larger tapes (Tape 48 and Sealing Tape, Cat. No. 236366) must also be carefully removed. The best way to do this is to peel off the tape as horizontally as possible so that the hard pulling on the Strips does not drag them out of the frame.

9. Inserting and removing NucleoLink Strips from thermal cyclers
The NucleoLink Strips are designed to fit into standard 0.2 ml thermal cycler blocks perfectly (

). As the red NucleoLink frames are not heat-stable, the Strips must be removed from the frame before adding them to the thermal cycler. The Strips are individually sealed with Tape 8 (Cat. No. 249719) (

), and the sealed Strips are inserted one after another into the thermal cycler (

). The individual sealing of NucleoLink Strips is necessary, because the Strips are inserted and removed individually from the thermal cycler.

After insertion of the NucleoLink Strips into the thermal cycler, the heated lid is closed. It is very important to assure that the lid is firmly closed and that the pressure is applied. Applying a firm and even pressure during thermal cycling prevents evaporation from the NucleoLink Strips.

9 a) Use of GeNunc Tape 48 during thermal cycling
For an experienced user it is possible to seal the NucleoLink Strips with thermostable Tape 48 (Cat. No. 232700) before inserting them into the thermal cycler. As the red NucleoLink frame is not heat-stable, it must be removed before inserting the Strips into the thermal cycler. Great care must be exercised when removing the Strips from the thermal block after thermal cycling. Since the wells fit into the block perfectly, the Strips are firmly attached to the block after thermal cycling, and there is a risk of breaking the Strips. For this reason the removal of the strips must be performed carefully in order not to break the Strips. This can be accomplished by means of a standard angle pincer (

), which is used to release the Strips very carefully by lifting them slightly (

). The Strips are then removed individually. When using Tape 48, the outermost Strips and wells should be very carefully released with the pincer, after which all the strips may be carefully lifted together from the thermal block.

9 b) Spacer Plate
A silicone Spacer Plate is placed on top of the NucleoLink Strips during thermal cycling to assure that the heated lid provides an evenly distributed pressure to the Strips (

). Furthermore, this assures that the rims of the single wells in the NucleoLink strips are under pressure, so the vapor pressure built up inside the wells during thermal cycling does not lift the tape and allow evaporation.

During thermal cycling, a small amount of glue from the tape will melt and adhere to the spacer plate. After prolonged use of the same spacer plate, the side facing the NucleoLink Strips will get sticky and may adhere to the Strips after thermal cycling. This can cause difficulties when the Spacer Plate is removed from the Strips, as the NucleoLink Strips will be lifted with the Spacer plate. Furthermore, if the Spacer Plate is turned so the side with the glue is facing the heated lid, the Spacer Plate may attach to the lid. The old Spacer Plate may remain attached to the heated lid. This may cause evaporation from a number of the wells during the next DIAPOPS analysis, if a new Spacer Plate is placed on the top of the NucleoLink Strips and the two Spacer Plates now used may create an uneven pressure on the Strips.

To remove the residual glue, the Spacer Plate should be regularly cleaned with ethanol.

10. ELISA readers
As the last step in the DIAPOPS procedure (

), the substrate reaction in the NucleoLink Strips is measured in an ELISA reader or in a fluorescence reader (

). If the Strips are measured in an ELISA reader, the OD value is measured by the loss of light intensity at a certain wavelength vertically through the well. This requires that the bottom is completely transparent. The NucleoLink Strips have flat bottoms that can be used with any wavelength. However, it may happen during the various washing steps that some of the high salt concentration buffer is spilled on the outside of the wells. This buffer can run down to the bottom and rest under the wells. During the incubation steps of the NucleoLink Strips, the plates are kept at elevated temperatures, and the liquid can evaporate. The precipitated salt, which is on the outside of the wells covering the bottom, can act as a shadow and stop the light. This will greatly affect the results. Inspection the bottom of the wells before they are measured in the ELISA reader is always recommended. Some fluorescent readers are not designed to measure through the bottom of the wells, and there should be no problem with these.

11. Can oil be used if the thermal cycler does not have a heated lid?
If the thermal cycler does not have a heated lid, or the lid is without pressure, it is possible to add one or two drops of oil to the NucleoLink wells after addition of the PCR mix. This will not affect the DIAPOPS results, if the oil is completely removed.

After thermal cycling, the liquid PCR mix is removed. If analysis of the liquid is not planned, the liquid and the oil are removed and discarded, as usual, and the wells are washed as described in the DIAPOPS procedure (

). If the liquid phase PCR product is to be analyzed, e.g. on an agarose gel (

), the liquid under the oil must be carefully removed using a pipette. No oil should be transferred to the gel with the PCR mix.

12. Conclusion
The NucleoLink Strips are handled much like MicroWell^® plates during normal ELISA. However, the washing step must be carefully conducted, as the sensitivity of DIAPOPS is very high (

). When washing the NucleoLink Strips, special washing equipment can be used with advantage, particularly when many plates are handled.