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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
- 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 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.
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 |
| iFluor™647-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.
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (260 nm) | Correction Factor (280 nm) |
| ReadiLink™ iFluor® 488 Nick Translation dsDNA Labeling Kit | 491 | 516 | 750001 | 0.91 | 0.21 | 0.11 |
| ReadiLink™ iFluor® 555 Nick Translation dsDNA Labeling Kit | 557 | 570 | 1000001 | 0.641 | 0.23 | 0.14 |
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
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
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 November 19, 2024
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