Amine-NHS Coupling Reaction

Prepare the solution of 1 mM amine-modified DNA in mili-Q grade water (10 µL), 1 M sodium bicarbonate in mili-Q grade water (1.5 µL), 10x PBS (1.5 µL), and NHS reagent in DMSO (2 µL)*.

Mix the reagent thoroughly and let them react for 30 – 60 min at room temperature (20–25 ºC). Add the mili-Q grade water (100 µL) and filter through P2 gel. The filtrates need to be purified by reversed-phase HPLC (0.1 M TEAA : acetonitrile).

* If the NHS-ester reagent contains hydrophobic moiety, such as DBCO and BHQ quenchers, please increase the DMSO concentration to 70%

Copper Catalyzed Azide-Alkyne Cyclization Reaction

Prepare the solution of 1 mM alkyne-modified DNA in mili-Q grade water (10 µL), 20 mM copper (II) sulfate in mili-Q grade water (10 µL), 40 mM THPTA ligand in water (10 µL), 100 mM sodium ascorbate in mili-Q grade water (2.5 µL), and azide reagent in DMSO (5 µL).

Mix the copper solution and THPTA solution and confirm the solution color turns blue. Then, add the sodium ascorbate solution, resulting in a colorless solution. Mix all solutions thoroughly and let them react for 1 hour at room temperature*.

* For some reagents, such as Tetrazine-PEG4-N3, requires higher DMSO concentration and 50 ºC incubation while reagents react.

Add the mili-Q grade water (100 µL) and filter through P2 gel. The filtrates need to be purified by reversed-phase HPLC (0.1 M TEAA : acetonitrile).