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

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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.

    Answer Id: E8083

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  • 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?

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    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.

    Answer Id: E8084

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    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?

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

    Answer Id: E8085

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

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    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.

    Answer Id: E8086

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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?

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

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

    Answer Id: E8087

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

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    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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    Is non-specific DNA added to the blocking buffer or hybridization buffers in the North2South Chemiluminescent Hybridization and Detection Kit?

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    Yes. The hybridization buffers for the North2South Chemiluminescent Hybridization and Detection Kit contain single stranded nucleic acids used to block the membrane during the pre-hybridization and hybridization procedures. The addition of more single stranded DNA or RNA is not needed.

    Answer Id: E8450

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

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    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.

    Answer Id: E8088

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    The nucleic acid probe is not fluorescent after labeling with ChromaTide™ nucleotides. What do you recommend I try?

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  • Check the base-to-dye ratio to determine the level of incorporation of the ChromaTide™ nucleotides. Since fluorescent detection may be affected by underlabeling, overlabeling, instrument sensitivity, or other factors, the base-to-dye ratio is a better indicator of incorporation efficiency.
  • ChromaTide™ nucleotides may not have been incorporated well in the enzymatic labeling reaction. Make sure that the enzymatic method used is compatible with the particular fluorescent ChromaTide™ nucleotide, since some methods may not be appropriate for all applications. You may also need to further optimize the enzymatic incorporation method, for example by optimizing enzyme concentration, incubation time, concentration, and ratio of labeled and unlabeled nucleotides. For PCR, a lower fidelity polymerase may give higher incorporation rates; however, incorporation rates will be generally low using PCR.
  • 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 fluorescent ChromaTide™ nucleotide 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: E8105

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  • Can I strip and re-probe a blot made used with the North2South Chemiluminescent Hybridization and Detection Kit?

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    Yes. The most common procedure for stripping DNA blots is to wash the blot 3 x 5 minutes with boiling 0.1% SDS. An additional alkaline wash of 3 x 5 minutes with 0.4 N NaOH, 0.1% SDS may also work for DNA blots. The blots must be neutralized in TE buffer after this wash method. RNA blots are best washed with 0.1 % SDS, 3 x 5 minutes, at 75-85°C. Commercial stripping buffers are also available. Stripped blots should be kept wet until re-probed by storage in TE buffer at 4°C. Blots may be stripped and re-probed several times.

    Answer Id: E8451

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

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    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.

    Answer Id: E8089

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    How long does the Qubit™ Fluorometer lamp last?

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    There are two light sources in the Qubit™ 2.0 Fluorometer. Both are LEDs. They are expected to last at least 5 years.

    Answer Id: E8123

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    I’m getting high background after labeling with ChromaTide™ nucleotides. What do you recommend I do?

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    You can try to purify the ChromaTide™ labeled probe with an appropriate spin column-based method to remove unincorporated ChromaTide™ nucleotides. Ethanol precipitation may not efficiently remove the unincorporated ChromaTide™ nucleotides, so a spin column will need to be used.

    Answer Id: E8106

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    Which method do you recommend using for transformation of Pichia?

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    We recommend electroporation for transformation of Pichia. Electroporation yields 10e3 to 10e4 transformants per μg of linearized DNA and does not destroy the cell wall of Pichia. If you do not have access to an electroporation device, you may use the Pichia Spheroplast Kit (Cat. No. K172001), PEG 1000 protocol (page 78 of the manual), LiCl protocol (page 80 of the manual), or the Pichia EasyComp™ Transformation Kit (Cat. No. K173001). We do not recommend spheroplasting for transformation of Pichia with plasmids containing an antibiotic resistance marker. Damage to the cell wall leads to increased sensitivity to the antibiotic, causing putative transformants to die before they express the antibiotic resistance gene. In contrast, spheroplasting can be used for transformation of PichiaPink™ vectors because these vectors are selected using auxotrophic markers.

    Answer Id: E9526

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