Protocol: Click-Chemistry Labeling of Oligonucleotides and DNA

Click chemistry is a versatile reaction that can be used for the synthesis of a variety of conjugates. Virtually any biomolecule can be easily labelled with small molecules, such as fluorescent dyes, biotin, etc using click chemistry method.

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 facilities and companies.

Protocol

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.

  1. 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
  2. Dissolve alkyne-modified oligonucleotide or DNA in water in a pressure-tight vial.
  3. Add 2M triethylammonium acetate buffer, pH 7.0, to final concentration 0.2 M.
  4. Add DMSO, and vortex.
  5. Add azide stock solution (10 mM in DMSO), and vortex.
  6. Add the required volume of 5mM Ascorbic Acid Stock solution to the mixture, and vortex briefly.
  7. Degass the solution by bubbling inert gas in it for 30 seconds. Nitrogen, argon, or helium can be used.
  8. 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.
  9. Vortex the mixture thoroughly. If significant precipitation of azide is observed, heat the vial for 3 minutes at 80°C, and vortex.
  10. Keep at room temperature overnight.
  11. 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.
  12. Centrifuge at 10000 rpm for 10 minutes.
  13. Discard the supernatant.
  14. Wash the pellet with acetone (1 mL), centrifuge at 10000 rpm for 10 minutes.
  15. 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.

Related products

BDP 558/568 azide

BDP 558/568 is a borondipyrromethene fluorophore with absorption and emission spectra close to Cyanine3. This is an azide derivative for сlick chemistry.
Show pricing
Cat. # Quantity Price Lead time
17430 1 mg $110.00 in stock
27430 5 mg $210.00 in stock
37430 10 mg $310.00 in stock
47430 25 mg $410.00 in stock
57430 50 mg $695.00 in stock
67430 100 mg please inquire in stock

BDP 650/665 alkyne

BDP 650/665 is a bright borondipyrromethene red emitting dye. The terminal ethynyl group of BDP 650/665 alkyne can be easily conjugated with various azides using a copper-catalyzed click reaction.
Show pricing
Cat. # Quantity Price Lead time
A84B0 1 mg $110.00 in stock
B84B0 5 mg $210.00 in stock
C84B0 10 mg $310.00 in stock
D84B0 25 mg $410.00 in stock
E84B0 50 mg $695.00 in stock
F84B0 100 mg $1190.00 5 days

BDP R6G alkyne

Terminal alkyne derivative of BDP R6G dye for Click chemistry. BDP R6G is a borondipyrromethene dye whose absorption and emission spectra are similar to R6G (rhodamine 6G).
Show pricing
Cat. # Quantity Price Lead time
A44B0 1 mg $110.00 in stock
B44B0 5 mg $210.00 in stock
C44B0 10 mg $310.00 in stock
D44B0 25 mg $410.00 in stock
E44B0 50 mg $695.00 in stock
F44B0 100 mg $1190.00 in stock

BDP TMR azide

Borondipyrromethene dye with azido group. Spectral properties are similar to TAMRA dye. Due to long fluorescence lifetime, BDP TMR is especially useful for fluorescence polarization measurements.
Show pricing
Cat. # Quantity Price Lead time
12430 1 mg $110.00 in stock
22430 5 mg $210.00 in stock
32430 10 mg $310.00 in stock
42430 25 mg $410.00 in stock
52430 50 mg $695.00 in stock
62430 100 mg $1190.00 in stock

Cyanine3.5 azide

Cyanine3.5 azide for biomolecule labeling through Click Chemistry.
Show pricing
Cat. # Quantity Price Lead time
12030 100 uL, 10 mM/DMSO $110.00 in stock
32030 500 uL, 10 mM/DMSO $210.00 in stock
42030 1 mL, 10 mM/DMSO $410.00 in stock
A2030 1 mg $110.00 in stock
B2030 5 mg $210.00 in stock
C2030 10 mg $310.00 in stock
D2030 25 mg $410.00 in stock
E2030 50 mg $695.00 in stock
F2030 100 mg $1190.00 in stock

Cyanine3 azide

Azide derivative of Cyanine3 fluorescent dye for сlick сhemistry.
Show pricing
Cat. # Quantity Price Lead time
11030 100 uL, 10 mM/DMSO $110.00 in stock
31030 500 uL, 10 mM/DMSO $210.00 in stock
41030 1 mL, 10 mM/DMSO $410.00 in stock
A1030 1 mg $110.00 in stock
B1030 5 mg $210.00 in stock
C1030 10 mg $310.00 in stock
D1030 25 mg $410.00 in stock
E1030 50 mg $695.00 in stock
F1030 100 mg $1190.00 in stock

Pyrene azide 1

Reagent for fluorescent labeling with pyrene polycyclic aromatic hydrocarbon via click chemistry.
Show pricing
Cat. # Quantity Price Lead time
A1530 1 mg $110.00 in stock
B1530 5 mg $210.00 in stock
C1530 10 mg $310.00 in stock
D1530 25 mg $410.00 in stock
E1530 50 mg $695.00 in stock

Pyrene azide 3

Pyrene azide with even longer linker for Click Chemistry.
Show pricing
Cat. # Quantity Price Lead time
NO items of this product available in stock but you can submit request for bulk.

R110 azide, 5- isomer

R110 dye azide, 5-isomer. R110 fluorescence is similar to FAM. This dye is azide for Click Chemistry.
Show pricing
Cat. # Quantity Price Lead time
NO items of this product available in stock but you can submit request for bulk.

R110 azide, 6- isomer

Rhodamine 110 (R110) azide for Click chemistry, 6-isomer.
Show pricing
Cat. # Quantity Price Lead time
15230 100 uL, 10 mM/DMSO $160.00 in stock
35230 500 uL, 10 mM/DMSO $420.00 in stock
45230 1 mL, 10 mM/DMSO $595.00 in stock
A5230 1 mg $110.00 in stock
D5230 25 mg $410.00 in stock
E5230 50 mg $695.00 5 days
F5230 100 mg please inquire 10 days

Sulfo-Cyanine7.5 azide

Sulfo-Cyanine7.5 is a very hydrophilic and water soluble dye for the near infrared region. The absorption and emission spectra of the dye are similar to indocyanine green (ICG), but sulfo-Cyanine7.5 has a much higher fluorescence quantum yield. The dye is a great choice for near infrared in vivo imaging. This is an azide for Click chemistry.
Show pricing
Cat. # Quantity Price Lead time
A6330 1 mg $110.00 in stock
B6330 5 mg $290.00 in stock
C6330 10 mg $470.00 in stock
D6330 25 mg $690.00 in stock
E6330 50 mg $1270.00 in stock
F6330 100 mg $1990.00 in stock

Your item has been added. View your cart or proceed to checkout
The count of items is incorrect.