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
- Prepare DNA samples
- Add reagents to tube
- Mix and centrifuge briefly
- Incubate at 15 °C for 60 minutes
- Place the reaction on ice followed by addition of Stop Solution and heating at 65 °C
- Place on ice for 5 minutes before using or store at 4 °C
- 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
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.
Table 1.Reagents composition per tube for each reaction
Components | Amount |
DNA sample | 1 µg DNA diluted in Nuclease-free water to final volume of 34 µL |
Nick Translation Buffer | 5 µL |
dNTP mix | 5 µL |
dTTP | 2 µL |
DIG-dUTP working solution | 2 µL |
DNA Polymerase I | 1 µL |
DNase I | 1 µL |
Total Volume | 50 µL |
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.
- 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.
- Carefully mix the reagents by a brief vortex followed by brief centrifuge.
- Incubate the reaction at 15 °C for 60 minutes.
- After incubation, place the reaction on ice.
- To terminate the reaction, add 5 µL of Stop Solution and heat the sample at 65 °C.
- Place on ice for 5 minutes before using or store at 4 °C.
- 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
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
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
Authors: Prato, C and Paper, T and Morinet, F
Journal: Journal of virological methods (1991): 227-31
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