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Labeling of Amino-Modified Oligonucleotides with Dye NHS Esters
Introduction
Reaction Setup:
Prepare a stock solution of 1 mg of the NHS-activated dye in 50 µL of anhydrous dimethyl formamide (DMF) or dimethyl sulfoxide (DMSO). 20 µL of this dye stock solution are added to a solution containing 20 - 30 nmol of the amino-modified oligonucleotide dissolved in 200 µL of a 50–100 mM bicarbonate buffer (pH 7.5 - 8.0). The mixture is allowed to stir for an additional 1–3 h at 25ºC.
Prepare a stock solution of 1 mg of the NHS-activated dye in 50 µL of anhydrous dimethyl formamide (DMF) or dimethyl sulfoxide (DMSO). 20 µL of this dye stock solution are added to a solution containing 20 - 30 nmol of the amino-modified oligonucleotide dissolved in 200 µL of a 50–100 mM bicarbonate buffer (pH 7.5 - 8.0). The mixture is allowed to stir for an additional 1–3 h at 25ºC.
Fig. 1
Labeling
Note:
- In most cases, the labeling reaction is completed within 30 minutes.
- It is critical that the solution used for labeling should be free of amines (TRIS buffer is not suitable as a labeling buffer for dye NHS-esters). Oligonucleotides stored in buffers containing amines need be dialyzed against the labeling buffer (phosphate-buffered saline (PBS), or sodium bicarbonate). In some instances it might be necessary to purify the oligonucleotide before labeling.
- Amino-modified oligo purification: 100 µg of the oligonucleotide are dissolved in 100 µl doubly distilled water and the solution is extracted three-times with chloroform and thereafter the oligonucleotide is precipitated by adding 20 µl of a 3 M sodium chloride solution and 250 µl of ethanol. The solution is mixed well and cooled for at least 30 min. at –20°C. After centrifugation for 20 - 30 min at 13,000 g, the supernatant is separated and discarded. The pellet is washed twice with cold ethanol (70%), dried (not entirely dry, otherwise it might be difficult to redissolve) and resolubilized in 150 - 250 µl Tris/HCl buffer, pH 8.0 or in distilled water.
Purification of the Dye-Oligonucleotide Conjugate:
The labeled oligonucleotide is first purified by repetitive ethanol precipitation as described above (see Note 3). The final purification is done by HPLC-separation on a reversed phase (C-18) column using an acetonitrile/H2O (80:20, v/v) gradient. Load the solution onto the column and run a linear solvent gradient of 0 - 75 % acetonitrile in water.
The labeled oligonucleotide is first purified by repetitive ethanol precipitation as described above (see Note 3). The final purification is done by HPLC-separation on a reversed phase (C-18) column using an acetonitrile/H2O (80:20, v/v) gradient. Load the solution onto the column and run a linear solvent gradient of 0 - 75 % acetonitrile in water.
Note:
- Purification of the labeled oligonucleotide may be achieved using commercially available quick separation columns following the instructions given by the supplier.
Storage of the Dye-Oligonucleotide Conjugates:
Dye-conjugates are to be stored under similar conditions as unlabeled oligonucleotids.
Dye-conjugates are to be stored under similar conditions as unlabeled oligonucleotids.
References
Oligonucletide Labeling Reagents, AAT Bioquest (2014).
Document: 02.0118.190605r1
Last updated Wed Oct 29 2025
Labeling of Amino-Modified Oligonucleotides with Dye NHS Esters