Dear Lakshani,

Please use the link in the following text:

1-Protocol: Click-Chemistry Labeling of Oligonucleotides and DNA

http://www.lumiprobe.com/protocols/click-chemistry-dna-labeling

Click chemistry is a versatile reaction that can be used for the synthesis of a variety of conjugates. Virtually any biomolecules can be involved, and labeling with small molecules, such as fluorescent dyes, biotin, and other groups can be readily achieved.

Click chemistry reaction takes place between two components: azide and alkyne (terminal acetylene). Both azido and alkyne groups are nearly never encountered in natural biomolecules. Hence, the reaction is highly bioorthogonal and specific. If there is a need to label an oligonucleotide, alkyne-modified oligonucleotides can be ordered at many of the custom oligo-synthesizing facilites and companies.

We recommend using the following general protocol for Click chemistry labeling of alkyne-modified oligonucleotides with azides produced by Lumiprobe Corp. The auxiliary reagents can be ordered at Lumiprobe Corp..

Calculate the volumes of reagents required for Click chemistry labeling using the table below. Prepare the required stock solutions (see Appendix).

Reagent            Final concentration in the mixture        Stock solution   concentration

Oligonucleotide, alkyne-modified  Varies (20 – 200 uM)               varies

Azide                 1.5 x (oligonucleotide concentration)                  10 mM in DMSO

DMSO                                50 vol %                                                                               -

Ascorbic acid                   0.5 mM                                                            5 mM in water

Cu-TBTA complex          0.5 mM                                           10 mM in 55 vol % DMSO

Dissolve alkyne-modified oligonucleotide or DNA in water in a pressure-tight vial.

Add 2M triethylammonium acetate buffer, pH 7.0, to final concentration 0.2 M.

Add DMSO, and vortex.

Add azide stock solution (10 mM in DMSO), and vortex.

Add the required volume of 5mM Ascorbic Acid Stock solution to the mixture, and vortex briefly.

Degass the solution by bubbling inert gas in it for 30 seconds. Nitrogen, argon, or helium can be used.

Add the required amount of 10 mM Copper (II)-TBTA Stock in 55% DMSO to the mixture. Flush the vial with inert gas and close the cap.

Vortex the mixture thoroughly. If significant precipitation of azide is observed, heat the vial for 3 minutes at 80°C, and vortex.

Keep at room temperature overnight.

Precipitate the conjugate with acetone (for oligonucleotides) or with ethanol (for DNA). Add at least 4-fold volume of acetone to the mixture (If the volume of the mixture is large, split in several vials). Mix thoroughly and keep at -20°C for 20 minutes.

Centrifuge at 10000 rpm for 10 minutes.

Discard the supernatant.

Wash the pellet with acetone (1 mL), centrifuge at 10000 rpm for 10 minutes.

Discard the supernatant, dry the pellet, and purify the conjugate by RP-HPLC or PAGE.

Appendix. Preparation of stock solutions of the reagents used for click-chemistry labeling and conjugation

5 mM Ascorbic Acid Stock

Preparation

Dissolve 18 mg of ascorbic acid in 20 mL of distilled water.

Storage

Ascorbic acid is readily oxidized by air. The solution is stable for one day. Use fresh preparations for Click chemistry.

10 mM Copper (II)-TBTA Stock in 55% DMSO

Preparation

Dissolve 50 mg of copper (II) sulfate pentahydrate in 10 mL of distilled water. Dissolve 116 mg of TBTA ligand in 11 mL of DMSO. Mix two solutions.

Storage

Store at room temperature. The solution is stable for years.

2M Triethylammonium Acetate Buffer, pH 7.0

Preparation

Mix 2.78 mL of triethylamine with 1.14 mL of acetic acid. Add water to 10 mL volume, and adjust pH to 7.0.

Storage

Store at room temperature. The solution is stable for years.

Hoping this will be helpful,

Rafik

Thank you Theo. I am using 6 FAM oligo modified with an azide. So I don't remove the excess DBCO since I use CY2 to visualize the DNA on PAGE. I am adding PBS as the buffer and let it to react at 37c overnight. On the gel I see  bands one  for unlabelled DNA one for the DBCO labelled DNA, and there is a band in the middle which I cannot recognize. 

Hi Theo, I am doing a competition reaction between DNA and DBCO labelled DNA on PAGE gel. Once DNA get labelled with DBCO it will move slowly so the band is shifted. To visualize the bands I need CY2 labelled oligo. Also the purpose of using CY5 labelled DBCO is for further in vivo  labeling. Anyway, band for the DBCO labelled DNA is very very weak.

Hi Lakshani,

After 2-6 hour reaction with DBCO-Cy5 (3-5 fold excess) you can easily remove excess DBCO-Cy5 by adding excess of Azide Agarose https://clickchemistrytools.com/product/azide-agarose/ .Incubate for 4-12 hours, and spin off agarose. This agarose contain azide groups that will react with excess of DBCO-Cy5, and will not react with your oligo.