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Invitrogen™ ARES™ Alexa Fluor™ 647 DNA Labeling Kit

Catalog No. A21676
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Description

ARES™ Alexa Fluor™ 647 DNA Labeling Kit

The ARES™ Alexa Fluor™ DNA Labeling Kit provides a versatile, two-step method for labeling DNA with our Alexa Fluor™ dyes. In the first step, an amine-modified nucleotide is incorporated into DNA using conventional enzymatic labeling methods. In the second step, the amine-modified DNA is chemically labeled using our proprietary amine-reactive Alexa Fluor™ 647 dye. The labeled probes can be used for fluorescence in situ hybridization (FISH) and microarray techniques. We offer ARES™ Alexa Fluor™ DNA Labeling Kits in five fluorescent colors, and each kit provides sufficient reagents for 5 to 10 labeling reactions of 1 to 5μg DNA each.

ARES™ Alexa Fluor™ Labeling Kit Specifications:
  • Dye (Ex/Em): Alexa Fluor™ 647 (650/670 nm)
  • Achieves more uniform, consistent labeling than techniques for enzymatic incorporation of labeled nucleotides
  • Typically produces one dye per 12–20 bases
  • Optimal for FISH and microarrays

    More Uniform Labeling With ARES™ Labeling Kits
    ARES™ Alexa Fluor™ Labeling Kits employ a two-step labeling technology—nick translation to enzymatically incorporate an amine-modified nucleotide (aminoallyl dUTP) followed by chemical labeling with Alexa Fluor™ dyes. This method achieves uniformity and consistency of labeling that is difficult to obtain with conventional enzymatic incorporation of labeled nucleotides.
    We also offer this two-step labeling technology in our FISH Tag™ DNA and FISH Tag™ RNA Kits, which provide a complete workflow solution for FISH applications, including all of the reagents for probe synthesis, labeling, purification and an anti-fade reagent to help protect the signal during fluorescence microscopy.

    More Options for Nucleic Acid Labeling
    To review various options for nucleic acid labeling (including DNA and RNA FISH), review Labeling Oligonucleotides and Nucleic Acids—Section 8.2 in the Molecular Probes™ Handbook.

    For Research Use Only. Not intended for human or animal therapeutic or diagnostic use.

    • Specifications

    Content And Storage Store in freezer (-5 to -30°C) and protect from light.
    Detection Method Fluorescence
    Format Kit
    Label or Dye Alexa Fluor 647
    Final Product Type Probes (Labeled DNA), cDNA (Labeled)
    Labeling Target DNA (General), cDNA
    Includes Label or Dye Yes
    Labeling Method Indirect Labeling
    Product Line ARES, Alexa Fluor
    Product Type DNA Labeling Kit
    Quantity 1 kit
    Shipping Condition Room Temperature
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    Frequently Asked Questions (FAQs)

    My purified RNA comes from multiple sources, but I am getting variable efficiency of labeling with the same ARES kit. What can cause this?

    Different preparations of RNA will certainly give different results. Most of the time, the mRNA is significantly degraded. The enzymatic incorporation of aminoallyl-dUTP (AA-dUTP) should not differ from reaction to reaction. If there are differences, it has to be due to the RNA or the method. AA-dUTP incorporation is no different than that of a dye-nucleotide conjugate, and should be more efficient and uniform. Here are a couple of suggestions:

    1) cDNA may have been lost prior to labeling. Add 1 µL of glycogen (molecular biology grade), containing 10-20 µg, to the cDNA before precipitating it with ethanol.

    2) Make sure to add sodium acetate as the salt and not ammonium acetate. After pelleting the cDNA, resuspend it in 5 µL water.

    3) If generating long cDNAs, it will help to heat-denature the sample. Heat it at 95°C for 5 minutes and then put it on ice for a few minutes. Then centrifuge it for a few minutes just prior to the labeling reaction.

    4) You want the tube to be at room temperature for the labeling reaction. Add the 3 µL of buffer and mix it in. Then add the dye and vortex it vigorously for at least 15 seconds.

    Could you make an ARES Alexa Fluor 633 DNA Labeling Kit? This would be a good fit with my 633 nm laser.

    Unfortunately, Alexa Fluor 633 does not label nucleic acids well because of the dye's chemical structure. Furthermore, DNA probes labeled with Alexa Fluor 633 do not form stable hybrids in nucleic acid hybridization assays.

    How stable is the ARES-labeled DNA to high temperature?

    An ARES-labeled oligonucleotide should survive at 95°C for 5 minutes.

    Can probes labeled using the ARES Alexa Fluor DNA Labeling Kits be stored for later use?

    Long-term storage for the ARES-labeled probes can be done in just about any kind of buffer, TE, formamide, hybridization buffer, or ethanol. We suggest using your normal storage conditions as long as you protect the probes from light. ARES conjugates are very stable.

    How do the Alexa Fluor dyes used in the ARES Alexa Fluor DNA Labeling Kits compare to Cy dyes for fluorescence intensity at different labeling ratios?

    At the same dye-to-base ratio, Alexa Fluor dyes exhibit higher intensity and reduced self-quenching at higher labeling ratios.

    What is the optimal degree of labeling (dye molecules per 100 base pairs) for labeled probes produced by the ARES Alexa Fluor DNA Labeling Kits?

    A dye-to-base ratio of 1:12 to 1:35 is a suitable range for Alexa Fluor dye labeling of cDNA for microarray and FISH (fluorescence in situ hybridization) applications.

    What can the ARES Alexa Fluor DNA Labeling Kits be used for?

    The ARES Alexa Fluor DNA Labeling Kits provides a versatile, two-step method for labeling DNA with our Alexa Fluor dyes. In the first step, an amine-modified nucleotide is incorporated into DNA using enzymatic labeling methods. In the second step, the amine-modified DNA is chemically labeled using our amine-reactive Alexa Fluor dyes. The labeled probes can be used for:

    - Fluorescence in situ hybridization (FISH). See Buster DW, Daniel SG, Nguyen HQ, et al. (2013) SCFSlimb ubiquitin ligase suppresses condensin II–mediated nuclear reorganization by degrading Cap-H2, J Cell Biol 201(1):49-63.
    - Microarray techniques. See Shin HH, Hwang BH, Seo JH et al. (2014) Specific discrimination of three pathogenic Salmonella enterica subsp. enterica serotypes by carB-based oligonucleotide microarray. Appl Environ Microbiol 80(1):366-373.

    For Research Use Only. Not for use in diagnostic procedures.