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ReadiLink™ Cy5 Nick Translation dsDNA Labeling Kit

ReadiLink™ Cy5 Nick Translation dsDNA Labelling Kit provides a simple and efficient way to label a double stranded DNA sample with the bright and photostable Cy5 dye. The labelling kit provides all necessary reagents for a complete workflow required for DNA labelling. This method utilizes a combination of DNAse and DNA polymerase to nick one strand of the DNA helix, to which Cy5 dye is conjugated. In addition, the kit allows the user to optimize incorporation and product size by adjusting the ratio of Cy5-dUTP conjugate to dTTP. It is compatible with a wide variety of sample materials, including bacterial artificial chromosome (BAC) DNA, human genomic DNA, purified PCR products, supercoiled and linearized plasmid DNA. The resulted Cy5-labeled DNAs can be used in a variety of molecular biology techniques such as fluorescence in situ hybridization (FISH).

Example protocol

AT A GLANCE

Protocol summary
  1. Prepare DNA samples
  2. Add reagents to tube
  3. Mix and centrifuge briefly
  4. Incubate at 15 °C for 60 minutes
  5. Place the reaction on ice followed by addition of Stop Solution and heating at 65 °C
  6. Place on ice for 5 minutes before using or store at 4 °C
  7. Purify the labelled DNA 

Important
Thaw all the kit components on ice before starting the experiment. Briefly vortex all the reagents to the bottom before starting the labelling process.

SAMPLE EXPERIMENTAL PROTOCOL

The following protocol can be used as a guideline.
Table 1.Reagents composition per tube for each reaction
ComponentsAmount
DNA sample1 µg DNA diluted in Nuclease-free water to final volume of 34 µL
Nick Translation Buffer5 µL
dNTP mix5 µL
dTTP2 µL
Cy5-dUTP working solution 2 µL
DNA Polymerase I1 µL
DNase I1 µL
Total Volume50 µL
The ratio of Cy5-dUTP (Component A): dTTP (Component E) can be optimized to achieve the best labelling conditions.
Incubation time can be optimized for better labelling. Longer incubation time will help with more labelling but may shorten the size of the end product.
  1. To a clean (Nuclease-free) 0.5 mL micro centrifuge tube or 0.2 mL PCR tube, add the reagents in the order indicated in Table 1.
  2. Carefully mix the reagents by a brief vortex followed by brief centrifuge.
  3. Incubate the reaction at 15 °C for 60 minutes.
  4. After incubation, place the reaction on ice.
  5. To terminate the reaction, add 5 µL of Stop Solution and heat the sample at 65 °C.
  6. Place on ice for 5 minutes before using or store at 4 °C.
  7. Purify the labeled DNA. 

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
ReadiLink™ Cy3 Nick Translation dsDNA Labeling Kit55556915000010.1510.070.073

References

View all 50 references: Citation Explorer
Customized optical mapping by CRISPR-Cas9 mediated DNA labeling with multiple sgRNAs.
Authors: Abid, Heba Z and Young, Eleanor and McCaffrey, Jennifer and Raseley, Kaitlin and Varapula, Dharma and Wang, Hung-Yi and Piazza, Danielle and Mell, Joshua and Xiao, Ming
Journal: Nucleic acids research (2021): e8
Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging.
Authors: O'Sullivan, Julia and Mersaoui, Sofiane Y and Poirier, Guy and Masson, Jean-Yves
Journal: Journal of visualized experiments : JoVE (2021)
Coupled DNA-labeling and sequencing approach enables the detection of viable-but-non-culturable Vibrio spp. in irrigation water sources in the Chesapeake Bay watershed.
Authors: Malayil, Leena and Chattopadhyay, Suhana and Mongodin, Emmanuel F and Sapkota, Amy R
Journal: Environmental microbiome (2021): 13
Fast and Efficient Postsynthetic DNA Labeling in Cells by Means of Strain-Promoted Sydnone-Alkyne Cycloadditions.
Authors: Krell, Katja and Pfeuffer, Bastian and Rönicke, Franziska and Chinoy, Zoeisha S and Favre, Camille and Friscourt, Frédéric and Wagenknecht, Hans-Achim
Journal: Chemistry (Weinheim an der Bergstrasse, Germany) (2021)
Metabolically-active bacteria in reclaimed water and ponds revealed using bromodeoxyuridine DNA labeling coupled with 16S rRNA and shotgun sequencing.
Authors: Malayil, Leena and Ramachandran, Padmini and Chattopadhyay, Suhana and Cagle, Robin and Hittle, Lauren and Ottesen, Andrea and Mongodin, Emmanuel F and Sapkota, Amy R
Journal: Water research (2020): 116185
Page updated on May 19, 2025

Ordering information

Price
Unit size
10 Reactions
20 Reactions
Catalog Number
1740817409
Quantity
Add to cart

Additional ordering information

Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
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Purchase orderSend to sales@aatbio.com
ShippingStandard overnight for United States, inquire for international
Request quotation

Spectral properties

Correction Factor (260 nm)

0.02

Correction Factor (280 nm)

0.03

Correction Factor (482 nm)

0.009

Correction Factor (565 nm)

0.09

Extinction coefficient (cm -1 M -1)

2500001

Excitation (nm)

651

Emission (nm)

670

Quantum yield

0.271, 0.42

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

Platform

Other instruments

Thermal Cycler

Components

Nick translation labeling of DNA starts with the creation of defects within the sequence of existing DNA double-helix molecules by cleavage of phosphodiester bonds with DNase along the backbone of one strand. Polymerase then repairs these nicks beginning with the removal of the adjacent nucleotide and the immediate filling back in of those gaps with new nucleotides from the added dNTP pool. As each new nucleotide is added, the polymerase leaves the 3′ OH group open, thus translating the nick toward the 5′ end. As the reaction sequence is repeated, the polymerase enzyme continues to remove existing nucleotides and replace them with new ones at the site of the new nick. The result of these reactions is numerous labeled and unlabeled nucleotides being incorporated as a complementary sequence along the length of each DNA strand, starting at the site of the original nick.
Nick translation labeling of DNA starts with the creation of defects within the sequence of existing DNA double-helix molecules by cleavage of phosphodiester bonds with DNase along the backbone of one strand. Polymerase then repairs these nicks beginning with the removal of the adjacent nucleotide and the immediate filling back in of those gaps with new nucleotides from the added dNTP pool. As each new nucleotide is added, the polymerase leaves the 3′ OH group open, thus translating the nick toward the 5′ end. As the reaction sequence is repeated, the polymerase enzyme continues to remove existing nucleotides and replace them with new ones at the site of the new nick. The result of these reactions is numerous labeled and unlabeled nucleotides being incorporated as a complementary sequence along the length of each DNA strand, starting at the site of the original nick.
Nick translation labeling of DNA starts with the creation of defects within the sequence of existing DNA double-helix molecules by cleavage of phosphodiester bonds with DNase along the backbone of one strand. Polymerase then repairs these nicks beginning with the removal of the adjacent nucleotide and the immediate filling back in of those gaps with new nucleotides from the added dNTP pool. As each new nucleotide is added, the polymerase leaves the 3′ OH group open, thus translating the nick toward the 5′ end. As the reaction sequence is repeated, the polymerase enzyme continues to remove existing nucleotides and replace them with new ones at the site of the new nick. The result of these reactions is numerous labeled and unlabeled nucleotides being incorporated as a complementary sequence along the length of each DNA strand, starting at the site of the original nick.

Direct upgrades

ReadiLink™ iFluor® 647 Nick Translation dsDNA Labeling Kit

Documents

pdfProduct protocol
pdfCertificate of analysis
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