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ReadiLink™ iFluor® 647 Nick Translation dsDNA Labeling Kit

ReadiLink™ iFluor® 647 Nick Translation dsDNA Labelling Kit provides a simple and efficient way to label a double stranded DNA sample with the bright and photostable iFluor® 647 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 iFluor 647 dye is conjugated. In addition, the kit allows the user to optimize incorporation and product size by adjusting the ratio of iFluor 647-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 iFluor® 647-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
iFluor™647-dUTP working solution 2 µL
DNA Polymerase I1 µL
DNase I1 µL
Total Volume50 µL
The ratio of iFluor™647-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™ iFluor® 488 Nick Translation dsDNA Labeling Kit4915167500010.910.210.11
ReadiLink™ iFluor® 555 Nick Translation dsDNA Labeling Kit55757010000010.6410.230.14

References

View all 2 references: Citation Explorer
Identification of bacterial cells by chromosomal painting.
Authors: Lanoil, B D and Giovannoni, S J
Journal: Applied and environmental microbiology (1997): 1118-23
Cyanine dye dUTP analogs for enzymatic labeling of DNA probes.
Authors: Yu, H and Chao, J and Patek, D and Mujumdar, R and Mujumdar, S and Waggoner, A S
Journal: Nucleic acids research (1994): 3226-32
Page updated on December 11, 2024

Ordering information

Price
Unit size
10 Reactions
20 Reactions
Catalog Number
1740417405
Quantity
Add to cart

Additional ordering information

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Request quotation

Spectral properties

Correction Factor (260 nm)

0.03

Correction Factor (280 nm)

0.03

Correction Factor (656 nm)

0.0793

Extinction coefficient (cm -1 M -1)

2500001

Excitation (nm)

656

Emission (nm)

670

Quantum yield

0.251

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.