1. Introduction
2. Routine handling of EDC
3. Comparison of EDC from different manufacturers
   1. A list of tested products
4. Storage of EDC
5. Conclusion

1. Introduction
EDC is a reagent frequently used in peptide synthesis ( ). The reagent is very active and the activated compounds are unstable in aqueous solution. EDC is therefore sensitive to moisture. It is important to handle the reagent under conditions which secures that it will remain active.

2. Routine handling of EDC
A good way of handling EDC is to separate it into aliquots immediately after opening the container for the first time. The standard procedure used at the Nunc A/S Research Laboratory is to divide the reagent into aliquots large enough for coating four or five NucleoLink plates with solid phase primer. Each aliquot contains about 100 mg EDC, as 21 mg is used for one plate. EDC aliquots are stored at -18ºC in Nunc CryoTube vials (Cat. No. 379146) in individual, sealed plastic bags containing a drying material. Before addition of EDC to the NucleoLink Strips, buffer is added directly to the CryoTube vials containing the reagent. The solution containing the dissolved EDC is transferred to a volume of buffer which is measured to obtain the correct final concentration of 10 mM. Following this, the phosphorylated solid phase primer is added to the EDC solution, and 100 µl of this mix is added to each well in the NucleoLink Strips.

3. Comparison of EDC from different manufacturers
In this study, ten different EDC products from seven different manufacturers were compared. The experiment was carried out with EDC taken directly from fresh containers to avoid any variation due to an in-house storage effect. Most EDC manufacturers suggested that the reagent should be stored either with desiccant material or under an atmosphere of argon or nitrogen. Furthermore, 8 out of 10 products should be stored cold, either at 4ºC or -20ºC. The remaining two products should be stored at a temperature below 15ºC or at room temperature (RT) respectively.

The comparison was based on the ability of EDC to mediate the binding of a 5'-end phosphorylated oligonucleotide to the surface of NucleoLink Strips. The amount of covalently bound solid phase oligonucleotide was measured using hybridization ( ). The coated NucleoLink Strips were treated at 94ºC for 20 minutes with 100 µl DIAPOPS wash buffer ( ) before hybridization, to assure that only thermostably bound oligonucleotides were measured.

Figure 1: The ability of different EDC products to covalently bind phosphorylated oligonucleotides to the surface of NucleoLink Strips, as a function of the coating time at 50ºC. A comparison between 10 products from 7 different producers was carried out. The number of solid phase primers is measured using hybridization with a labelled probe ( ).

The results presented in Figure 1 indicate that there is no significant difference between the large majority of products. The only exception is an EDC from a producer who suggested that the reagent should be stored at RT. This reagent yields very low binding of the solid phase oligonucleotide, whereas there is no real difference between the other products. However, this product is a methiodide salt, and for this reason the concentration could be suboptimal, as the concentration of 10 mM ( ) is optimized for the hydrochloride salt of EDC.

3 a). A list of the tested products
Table 1 shows the different producers and the products from these producers that were tested. The name of EDC is given for each producer, because there are many different names in use. This difference is mainly dependent upon the country of the manufacturer.

Table 1: Presenting the tested EDC products with producers, name variants, and catalogue numbers.

4. Storage of EDC
To evaluate how important the storage temperature is for EDC, 3 different aliquots of our standard in-house reference EDC (Sigma E 7750) were stored without desiccant material at different temperatures for one month and compared with the standard storage method; at -20ºC with drying material. The results are presented in Figure 2. It is obvious that there is a difference in the EDC coating efficiency after storage for one month as a function of the temperature at which the reagent was stored. Thus, the storage conditions for EDC should be controlled very carefully. If there is any doubt whether the EDC has been stored correctly, a new shipment should be purchased immediately as a relatively short time under the wrong storage conditions could reduce the condensing ability of EDC.

Figure 2: A comparison of the ability of EDC, stored at different temperatures, to covalently bind a phosphorylated oligonucleotide to the NucleoLink surface. The solid phase oligonucleotides were measured using hybridization ( ).

 5. Conclusion
A comparison of ten different EDC products showed that there was no large difference in the efficiency to bind a phosphorylated oligonucleotide to the surface of the NucleoLink Strips. Only one product gave a lower result. The storage conditions of EDC are very important. After only one month, a difference could be seen, depending on the storage temperature.