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ReadiLink™ DIG (Digoxigenin) Nick Translation dsDNA Labeling Kit

ReadiLink™ DIG Nick Translation dsDNA Labelling Kit provides a simple and efficient way to label a double stranded DNA sample with DIG tag. 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 DIG is conjugated. In addition, the kit allows the user to optimize incorporation and product size by adjusting the ratio of DIG-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 DIG-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
DIG-dUTP working solution 2 µL
DNA Polymerase I1 µL
DNase I1 µL
Total Volume50 µL
The ratio of DIG-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. 

Citations

View all 1 citations: Citation Explorer
LncRNA AL592284. 1 facilitates proliferation and metastasis of cervical cancer cells via miR-30a-5p/Vimentin/EMT axis
Authors: Zhang, Jing and Liu, Hong-li and Liu, Jing-bo and Zhang, Yuan and Liu, Jing and Li, Yan-hua
Journal: Biochemical and Biophysical Research Communications (2021): 95--102

References

View all 2 references: Citation Explorer
[In situ hybridization of cells infected by Chlamydia trachomatis].
Authors: Mortemousque, B and Verin, P and Bebear, C and de Barbeyrac, B and Gendre, P
Journal: Revue internationale du trachome et de pathologie oculaire tropicale et subtropicale et de sante publique : organe de la Ligue c (1994): 47-62
Use of M13 single-stranded DNA digoxigenin labelled probe for detection of human parvovirus B19 viraemia.
Authors: Prato, C and Paper, T and Morinet, F
Journal: Journal of virological methods (1991): 227-31
Page updated on December 17, 2024

Ordering information

Price
Unit size
10 Reactions
20 Reactions
Catalog Number
1747217473
Quantity
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Additional ordering information

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

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.
Verification of double stranded DNA labeling with ReadiLink™ DIG (Digoxigenin) Nick Translation dsDNA Labeling Kit. Double stranded DNA were labeled with ReadiLink™ DIG (Digoxigenin) Nick Translation dsDNA Labeling Kit, purified and then incubated with or without anti-DIG antibody before applied to agarose gel electrophoresis. Gel was stained with Gelite™ Safe DNA Gel Stain. Anti-DIG antibody caused a supershift of DIG-labeled dsDNA while in the absence of antibody, shift was not obseved.

Documents

pdfSafety data sheet
pdfProduct protocol
pdfCertificate of analysis
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