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Helixyte™ iFluor® 350 Nucleic Acid Labeling Dye *Optimized for Labeling 2x100 ug DNA/RNA*

Helixyte™ iFluor® 350 Nucleic Acid Labeling Dye is a key member of our enabling Helixyte™ nucleic acid labeling and conjugation technology. The labeling/conjugation of a tag/hapten to nucleic acids has been very challenging due to the lack of reactive moieties in nucleic acid molecules. Thymine and guanosine have been often explored for nucleic acid conjugations, e.g., photo-crosslink (to thymine by psoralens) or bromination/Ulysis labeling of guanosine. However, these existing conjugations are either tedious or require stringent conditions with low yields and are thus not suitable for routine lab use. Under the similar conditions, our Helixyte™ nucleic acid labeling and conjugation technology is much easier to use with significantly higher yield. Helixyte™ iFluor® 350 Nucleic Acid Labeling Dye provides a unique method to attach the iFluor® 350 fluorophore to nucleic acids via a simple mixing step. The labeling reagent readily reacts with the N7 of guanine to form a stable covalent bond. The labeling procedure is simple and fast with a high production yield. The separation of the labeled nucleic acids from the unreacted dye can be accomplished with a simple ethanol precipitation, a spin-column or dialysis. The resulting labeled DNA/RNA probes have bright blue fluorescence that can be easily detected with AMCA/Alexa Fluor 350 filter set. They can be used for dot, Northern and Southern blots, RNA and DNA in situ hybridization, multicolor fluorescence in situ hybridization (mFISH), comparative genome hybridization (CGH) or microarray analysis etc.

Example protocol

AT A GLANCE

Protocol Summary
  1. Combine DNA with the Helixyte™ iFluor® 350 Nucleic Acid Labeling Dye stock solution.

  2. Incubate for 1 hour at 37°C.

  3. Purify the conjugate as required for downstream applications.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Important

Before opening the vial, thaw Helixyte™ iFluor® nucleic acid labeling dye at room temperature. Briefly centrifuge to collect the dried pellet.

Prepare a Helixyte™ iFluor® Nucleic Acid Dye Stock Solution
  1. Add 110 μL of DMSO to the Helixyte™ iFluor® 350 Nucleic Acid Labeling Dye vial to prepare a 10 mM stock solution.

    Note: It is recommended to divide any unused stock solution into single-use aliquots. Store the aliquots at ≤-20 ºC and protect them from light. Avoid repeated freeze-thaw cycles.

SAMPLE EXPERIMENTAL PROTOCOL

Protocol
  1. Prepare the labeling reaction according to the specifications in table 1 below.

    Table 1. Standard Nucleic Acid Labeling Reaction.

    ComponentsVolume added to reactionFinal Concentration
    DNA (1 mg/mL)2 to 5 µL2 to 5 µg
    Helixyte™ iFluor® 350 Nucleic acid Labeling Dye stock solution0.5 µL50 µM
    TE Buffer (pH 8 to 8.5)Add sufficient buffer to adjust the volume to 100 µL 

     

    Note: This DNA:Dye ratio results in labeling efficiencies that are appropriate for most applications. The amount of Helixyte™ iFluor® 350 Nucleic Acid Labeling Dye or the reaction incubation time can be adjusted to modify the labeling density as per the application requirements. The DNA-to-dye ratio must be optimized to achieve a higher labeling ratio

  2. Incubate the reaction at 37℃ for 1 hour, protected from light.

    Note: After 30 minutes of incubation, briefly centrifuge the reaction to minimize the effects of evaporation and maintain the appropriate concentration of the reaction components.

    Note: Alternatively, the reaction can be incubated at room temperature for 2 hours. For the best labeling condition, we recommend incubating at 37℃.

  3. After incubation, the labeling mix can be purified to remove any free labeling dye. Refer to the “Purification of labeling mix with alcohol precipitation” section below for instructions.

Purification of Labeling Mix with Alcohol Precipitation
  1. Add 0.1 volume (10 uL) of 5M sodium chloride and 2 - 2.5 volumes of ice-cold 100% ethanol (250 uL) to the reaction. Mix well and place at ≤ -20°C for at least 30 minutes.

  2. Centrifuge at full speed (>14,000 x g) in a refrigerated micro centrifuge for 15-30 minutes to pellet the labeled nucleic acid. Once pelleted, carefully remove the ethanol with a micropipette. Do not disturb the pellet.

    Note: Small nucleic acid quantities can be difficult to visualize. Mark and orient the precipitate-containing tubes in the microfuge such that the pellet will form in a predetermined place.

  3. Wash the pellet once with 500 μL of room temperature 70% ethanol. Centrifuge at full speed for an additional 15-30 minutes.

  4. Remove all traces of ethanol with a micropipette. DO NOT allow the sample to dry longer than 5 minutes as the pellet may become difficult to resuspend.

  5. Resuspend the labeled DNA with ~ 30 µL sterile water.

  6. Store the purified, labeled nucleic acid for long-term storage or put on ice for immediate use.

Spectrum

References

View all 50 references: Citation Explorer
Visualizing DNA replication by single-molecule analysis of replicated DNA.
Authors: Madireddy, Advaitha and Gerhardt, Jeannine
Journal: STAR protocols (2023): 102721
Glass Nanopipette-Based Plasmonic SERS Platform for Single-Cell MicroRNA-21 Sensing during Apoptosis.
Authors: Wang, Yong and Wang, Dandan and Qi, Guohua and Hu, Ping and Wang, Erkang and Jin, Yongdong
Journal: Analytical chemistry (2023): 16234-16242
Rapid, inexpensive, sequence-independent fluorescent labeling of phosphorothioate DNA.
Authors: Satusky, Matthew J and Johnson, Caitlin V and Erie, Dorothy A
Journal: Biophysical journal (2023): 1211-1218
A Step toward Amino Acid-Labeled DNA Sequencing: Boosting Transmission Sensitivity of Graphene Nanogap.
Authors: Mittal, Sneha and Pathak, Biswarup
Journal: ACS applied bio materials (2023): 218-227
Inhibition of N-Acetyltransferase 10 Suppresses the Progression of Prostate Cancer through Regulation of DNA Replication.
Authors: Ma, Ningning and Liu, Haijing and Wu, Yaqian and Yao, Mengfei and Zhang, Bo
Journal: International journal of molecular sciences (2022)
Page updated on December 6, 2024

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Catalog Number17950
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Physical properties

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.83

Correction Factor (280 nm)

0.23

Extinction coefficient (cm -1 M -1)

200001

Excitation (nm)

345

Emission (nm)

450

Quantum yield

0.951

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
Direct labeling of nucleic acid using Helixyte™ iFluor® 350 Nucleic Acid Labeling Dye. DNA ladder was labeled with 50 µM of Helixyte™ iFluor® 350 Nucleic Acid Labeling Dye (Lane 4) and analyzed alongside unlabeled DNA (Lane 1) on 1% agarose DNA gel using gel electrophoresis.
Direct labeling of nucleic acid using Helixyte™ iFluor® 350 Nucleic Acid Labeling Dye. DNA ladder was labeled with 50 µM of Helixyte™ iFluor® 350 Nucleic Acid Labeling Dye (Lane 4) and analyzed alongside unlabeled DNA (Lane 1) on 1% agarose DNA gel using gel electrophoresis.
Direct labeling of nucleic acid using Helixyte™ iFluor® 350 Nucleic Acid Labeling Dye. DNA ladder was labeled with 50 µM of Helixyte™ iFluor® 350 Nucleic Acid Labeling Dye (Lane 4) and analyzed alongside unlabeled DNA (Lane 1) on 1% agarose DNA gel using gel electrophoresis.