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RISH Rembrandt Biotin and Digoxigenin-Labeled RNA Probes Manual / Product Insert

  • Version: 24 Dec 2013
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What size of amine-modified oligonucleotides can be used with Alexa Fluor Oligonucleotide Amine Labeling Kits? Product FAQ

Answer

The kits have been optimized for labeling 50 µg of a 5'-amine-modified oligonucleotide that is 18 to 24 bases in length per reaction. Slightly shorter or longer oligonucleotides may be labeled by the same procedure; however, you may need to adjustment the protocol for greatly shorter or longer oligonucleotides. We have not tested the kits with oligonucleotides containing more than one amine.

Answer Id: E8071

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How do I obtain an amine-modified oligonucleotide for labeling using the Alexa Fluor Oligonucleotide Amine Labeling Kits? Product FAQ

Answer

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).

Answer Id: E8075

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How much amine-reactive Alexa Fluor dye is included in the Alexa Fluor Oligonucleotide Amine Labeling Kits? Product FAQ

Answer

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.

Answer Id: E8073

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I'm getting high background when using the AlexaFluor Oligonucleotide Amine Labeling Kit. What could be the cause of this? Product FAQ

Answer

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.

Answer Id: E8104

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My oligonucleotide is not fluorescent after the labeling reaction, and/or the labeling reaction did not work. What could be the cause of this? Product FAQ

Answer

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.

Answer Id: E8103

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Application Note: Nucleic Acid Therapeutics Applications Notebook Product Literature

How do I purify the labeled oligonucleotide from the unreacted dye and unlabeled oligonucleotide following the Alexa Fluor Oligonucleotide Amine Labeling reaction? Product FAQ

Answer

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).

Answer Id: E8074

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What dyes are used in the Alexa Fluor Oligonucleotide Amine Labeling Kits? Product FAQ

Answer

The kits include our Alexa Fluor amine-reactive succinimidyl or tetrafluorophenyl (TFP) ester dyes, which can be used for covalent conjugation to a primary amine.

Answer Id: E8072

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Alexa Fluor Oligonucleotide Amine Labeling Kits Manual / Product Insert

  • Version: 02-01-2006
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Alexa Fluor Oligonucleotide Amine Labeling Kits Quick Reference Manual / Product Insert

  • Version: 27 August 2012
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REMBRANDT Biotin and Digoxigenin-labeled DNA and RNA Control Probes User Guide Manual / Product Insert

  • Version: 24 Dec 2013
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How do I reconstitute my lyophilized oligonucleotide? How long can I store the oligonucleotide? Product FAQ

Answer

Centrifuge the tube for a few seconds to collect the oligonucleotide at the bottom of the tube. Carefully open the tube, and dissolve the oligonucleotide in the appropriate volume of TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). TE is recommended over deionized water since the pH of water is often slightly acidic and can cause hydrolysis of the oligonucleotide. It is also best to pipette the solution up and down at least 10 times. Please visit this webpage (https://www.thermofisher.com/us/en/home/life-science/oligonucleotides-primers-probes-genes/custom-dna-oligos/oligo-technical-resources/oligo-protocols.html) for more information on how to calculate primer concentration and resuspension volume.

The lyophilized oligonucleotide is stable at -20 degrees C for 1 year or more. The oligonucleotide dissolved in TE is stable for at least 6 months at -20 degrees C or 4 degrees C. Oligonucleotides dissolved in water are stable for at least 6 months at -20 degrees C. Do not store oligonucleotides in water at 4 degrees C.

Answer Id: E2937

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How do I determine the percentage of full-length oligonucleotide? Product FAQ

Answer

The percentage of full-length oligonucleotide depends on the coupling efficiency of the chemical synthesis. The average efficiency is close to 99%. To calculate the percentage of full-length oligonucleotide, use the formula: 0.99n-1. Therefore, 79% of the oligonucleotide molecules in the tube are 25-bases long; the rest are <25 bases. If you are concerned about starting with a preparation of oligonucleotide that is full-length you may want to consider cartridge, PAGE, or HPLC purification.

Answer Id: E7279

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Do I really need to include a control when doing siRNA transfection? Product FAQ

Answer

It is absolutely critical to have a control oligonucleotide to be able to determine any non-specific effects. This oligonucleotide can be a “scrambled” oligonucleotide (same length and base composition in a random order) or a “sense” oligonucleotide if the target is mRNA.

Answer Id: E3850

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