Alexa Fluor™ 488 Oligonucleotide Amine Labeling Kit

Insufficient removal of free dye could lead to high background. Try purifying the oligonucleotides by HPLC or gel electrophoresis to ensure removal of unreacted dye.

Here are some possibilities and our suggestions for addressing them:

- Check the age of the Alexa Fluor amine-reactive dye and how it has been stored. The dyes are sensitive to hydrolysis and can lose reactivity if exposed to moisture or water. They should be stored as a powder and dessicated to protect the dye from water. Anhydrous DMSO should be used for dissolving the dye, and once the dye is dissolved in DMSO, it should be used right away, since it will be more sensitive to hydrolysis and less stable in solution. The reactive dye should also be protected from light during storage. When stored dessicated and protected from light in powder form, dyes are stable for at least 6 months; however, dyes older than this may have hydrolyzed and no longer be reactive.
- For some reactions, a larger dye-to-oligonucleotide molar ratio may be necessary, so you may need to use more dye or less oligonucleotide in the reaction.
- The reaction works best at a slightly basic pH so that the amine on the oligonucleotide is deprotonated, so a 0.1 M sodium borate, pH 8.5 labeling buffer should be used for the reaction. For best results, other buffers should not be used, since they may not have the correct pH or may contain components that interfere with the reaction.
- Make sure there are no proteins or primary amines in the reaction. The amine-modified oligonucleotide should be extracted and purified before the reaction to remove any amines such as Tris, triethylamine, ammonium salts, glycine, BSA, or other amine-containing molecules since these will react with the amine-reactive dye and reduce the efficiency of the reaction.
- Check the fluorescent filter used for detection to make sure it is compatible with the dye. You can also test a small drop of the undiluted dye in your filter to make sure you can image the dye alone before it is conjugated to the oligonucleotide. The fluorescence emission of Alexa Fluor 647 is not visible by eye and will require a far-red imaging system for detection.

An amine-modified oligonucleotide can be obtained from commercial suppliers of custom oligonucleotides. For information about our custom DNA oligo service, go here (https://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos.html).

We recommend gel electrophoresis or reverse-phase HPLC after an ethanol precipitation step. More details on the purification procedure can be found in the product manual (http://tools.thermofisher.com/content/sfs/manuals/mp20191.pdf).

The exact amount of reactive dye per vial is proprietary; however, each vial provides an optimal amount of dye for labeling 50 µg of a 5'-amine-modified oligonucleotide that is 18 to 24 bases in length. The kit includes 3 vials of dye for 3 reactions total. The amine-reactive Alexa Fluor dyes can also be purchased as stand-alone reagents in amounts ranging from 100 µg to 25 mg.