ReadiLink™ iFluor® 555 Nick Translation dsDNA Labeling Kit

ReadiLink™ iFluor® 555 Nick Translation dsDNA Labelling Kit provides a simple and efficient way to label a double stranded DNA sample with the bright and photostable iFluor® 555 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 555 dye is conjugated. In addition, the kit allows the user to optimize incorporation and product size by adjusting the ratio of iFluor 555-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® 555-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

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™555-dUTP working solution	2 µL
DNA Polymerase I	1 µL
DNase I	1 µL
Total Volume	50 µL

The ratio of iFluor™555-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.

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

Open in Advanced Spectrum Viewer

Product family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)	Correction Factor (656 nm)
ReadiLink™ iFluor® 488 Nick Translation dsDNA Labeling Kit	491	516	75000¹	0.9¹	0.21	0.11	-
ReadiLink™ iFluor® 647 Nick Translation dsDNA Labeling Kit	656	670	250000¹	0.25¹	0.03	0.03	0.0793

References

View all 6 references: Citation Explorer

Engineering nicking enzymes that preferentially nick 5-methylcytosine-modified DNA.
Authors: Gutjahr, Alice and Xu, Shuang-yong
Journal: Nucleic acids research (2014): e77

Pro-apoptotic gene knockdown mediated by nanocomplexed siRNA reduces radiation damage in primary salivary gland cultures.
Authors: Arany, Szilvia and Xu, Qingfu and Hernady, Eric and Benoit, Danielle S W and Dewhurst, Steve and Ovitt, Catherine E
Journal: Journal of cellular biochemistry (2012): 1955-65

Identification of bacterial cells by chromosomal painting.
Authors: Lanoil, B D and Giovannoni, S J
Journal: Applied and environmental microbiology (1997): 1118-23

An evaluation of a new series of fluorescent dUTPs for fluorescence in situ hybridization.
Authors: Wiegant, J and Verwoerd, N and Mascheretti, S and Bolk, M and Tanke, H J and Raap, A K
Journal: The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society (1996): 525-9

Directly labeled DNA probes using fluorescent nucleotides with different length linkers.
Authors: Zhu, Z and Chao, J and Yu, H and Waggoner, A S
Journal: Nucleic acids research (1994): 3418-22

Page updated on December 17, 2024

Ordering information

Price
Unit size	10 Reactions 20 Reactions
Catalog Number	1740217403
Quantity

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Additional ordering information

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Spectral properties

Correction Factor (260 nm)	0.23
Correction Factor (280 nm)	0.14
Extinction coefficient (cm ^-1 M ^-1)	100000¹
Excitation (nm)	557
Emission (nm)	570
Quantum yield	0.64¹