AAT Bioquest

What are the general procedures for click chemistry labeling of oligonucleotide and DNA?

Posted April 10, 2024


The general procedure for click chemistry labeling of oligonucleotide and DNA is described in the steps below. 

  1. Prepare Oligonucleotide Solution: Dissolve the alkyne-modified oligonucleotide or DNA in water into a vial. Add 2M triethylammonium acetate buffer (pH 7.0) to reach a final concentration of 0.2 M. Mix in DMSO and vortex the solution.
  2. Add Azide: Mix in the azide stock solution (10 mM in DMSO) and vortex again. 
  3. Add Ascorbic Acid: Add the required volume of 5 mM Ascorbic Acid stock solution and briefly vortex. 
  4. Degassing: Bubble inert gas (nitrogen, argon, or helium) into the solution for 30 seconds to degas it. 
  5. Add Cu-TBTA Complex: Add the required amount of 10 mM Copper (II)-TBTA stock solution in 55% DMSO. Flush the vial with inert gas and close the cap. Vortex the mixture, if precipitation occurs, heat the vial at 80°C for 3 minutes and then vortex again. 
  6. Incubation: Allow the mixture to incubate at room temperature overnight.
  7. Precipitation: For oligonucleotide conjugates: Add at least a 4-fold excess volume of 3% lithium perchlorate in acetone. For DNA conjugates: Add sodium acetate to reach a final concentration of 0.3 M, then add 2.5 volumes of ethanol (or 0.8 volumes of isopropanol). Mix and let it sit at −20 °C for 20 minutes.
  8. Centrifugation and discarding: Centrifuge at 10000 rpm for 10 minutes and discard the supernatant. Then wash the pellet with acetone (1 mL) and centrifuge again at 10000 rpm for 10 minutes. Discard the supernatant again, dry the pellet, and purify the conjugate via PAGE or RP-HPL.
Additional resources

A Hitchhiker’s Guide to Click-Chemistry with Nucleic Acids

Click Chemistry

FastClick™ Cy3 Azide