Preparation of Bromohexyl-modified oligonucleotides for HaloTag conjugation

Figure 1: Structures of Spacer 18 and 5'-Bromohexyl Phosphoramidites


Spacer 18



To prevent elimination or displacement of the bromine, monomers that allow for UltraMILD deprotection must be used. (dA: 10-1601-xx, dC: 10-1015-xx, dG: 10-1621-xx, dT: 10-1030-xx ). To avoid any exchange of the iPr-Pac group on the dG with acetyl, use the UltraMild Cap Mix A (40-4210-xx/ 40-4212-xx). Both the 5'-Bromohexyl and Spacer Phosphoramidite 18 are oils that require 5-10 minutes with occasional shaking to go fully into solution. A 3 minute coupling time is recommended for the 5'-Bromohexyl Phosphoramidite.


Fit the synthesis column with two syringes and deprotect using 1 mL of 50 mM Potassium Carbonate in methanol for 4 hours at room temperature. Transfer the cleaved oligo solution into a clean vial and rinse the support with an additional 1 mL of 50 mM Potassium Carbonate in methanol and combine. Neutralize the solution using 12µL glacial acetic acid and dilute with an equal volume of 2 M TEAA, pH 7.0. Dilute to 10 mL and desalt using a Glen-Pak DNA cartridge.


See DNA conjugation to Halotagged Protein using Glen Bromohexyl Linker