Applicable Products

General

Although Ac-dC-CE Phosphoramidite (LK2034) can be deprotected under UltraMILD conditions this monomer is routinely used under standard and UltraFAST conditions. Customers should refer to the standard protocols for use of this product and the analogous CPG supports.

Physical & Dilution Data

Dilution volumes (in ml) are for 0.1M solutions in dry acetonitrile (LK4050). Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.

Coupling

No changes are required from the standard synthesiser procedure. Likewise, supports are be treated as normal. If many dG residues are included in the oligonucleotide, we recommend the use of phenoxyacetic anhydride in Cap Mix A (LK4210). This removes the possibility of exchange of the iPr-Pac protecting group on the dG moiety with acetate from the standard acetic anhydride capping mix.

Deprotection & Purification

We no longer stock UltraMILD Deprotection solution (0.05M Potassium Carbonate in Methanol). This is because in our experience this reagent works best when freshly prepared just prior to use. To prepare 100ml of such a solution:

1. To a suitable volumetric calibrated vessel, add 0.69g potassium carbonate (K2CO3).
2. Add dry methanol to a volume of 100ml.
3. Stir, protected from moisture, until dissolved leaving overnight if necessary.

Cleavage and deprotection can be carried out in 4-17h at room temperature with this solution (alternatively use AMA at room temperature for 2h; ultimately the overall protocol will be dependent on the requirements of the base-sensitive modifications being used). Cartridge purification can be incorporated into the deprotection protocol.

Typical Protocol

1. Carry out the synthesis of the modified oligonucleotide.
2. Open the synthesis column and transfer the support to a suitable reaction vial.
3. Treat the support with 0.5ml of 0.05M potassium carbonate in anhydrous methanol for a minimum of 4h at room temperature. For oligos with a high dG content reaction overnight is recommended.
4. Desalt with a G25 column.

Storage & Stability

Phosphoramidites and supports are stored refrigerated at 2 to 8°C. Phosphoramidite solutions should be used within 24h.