ReadiLink™ iFluor® 647 FISH Fluorescence Imaging Kit

Telomere quantitative fluorescence in situ hybridization in metaphase HeLa cells using iFluor® 647-dUTP labeled telomere probes. Probes were created using the ReadiLink™ iFluor® 647 FISH Fluorescence Imaging kit.

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
Shipping	Standard overnight for United States, inquire for international

Physical properties

Solvent

Water

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-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure

Overview SDS Protocol

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

Fluorescence in situ hybridization (FISH) technology is an effective tool for detecting specific nucleic acid targets in a biological specimen. Detection of a nucleic acid target in situ is achieved through the hybridization of a fluorescent dye-labeled nucleic acid probe of complementary sequence to the specimen. The Readilink™ iFluor® 647 FISH fluorescence imaging kit is a convenient tool for labeling a target DNA using an iFluor® 647 labeled FISH probe via in situ hybridization. The kit provides Taq DNA polymerase enzyme which incorporates iFluor® 647-dUTPs in the target DNA through Polymerase Chain Reaction (PCR). Our proprietary iFluor® dyes are brighter and more photostable than traditional fluorescent labels, providing the desired resolution and signal.

Platform

Thermal Cycler

Recommended plate

PCR Microplate

Components

Example protocol

SAMPLE EXPERIMENTAL PROTOCOL

Before using, thaw all components to room temperature and mix thoroughly by vortexing.

Note: The following protocol can be used as a general guideline to standard DNA FISH. Optimization may be necessary for your experimental system.

Prepare the following reaction mixes as indicated in Table 1.

Table 1. Reagents composition per well for each reaction.

Components	Volume (25 µL/reaction)	Final Conc.
FISH Reaction mix (2X)	12.5 µL	1X
Upstream primer, 10 µM	0.25-2.5 µL	0.1-1.0 µM
Downstream primer, 10 µM	0.25-2.5 µL	0.1-1.0 µM
DNA template	1-5 µL	Optimized conc.
iFluor® 647-dUTP	2.5 µL
dNTP mix	1 µL
Water, nuclease-free	25 µL

Carefully mix the reagents by gentle vortexing followed by a brief centrifuge.
Set up the plate in the qPCR instrument and run as indicated in Table 2.

Table 2. Thermal cycling parameters.

Parameter

Polymerase Activation

PCR (30-40 cycles)

Hold

Denature

Anneal

Extend

Temperature

95 °C

95 °C

55-65 °C

68-72 °C

Time (m:ss)

0:20

0:30

1:00

1:00

Spectrum

Open in Advanced Spectrum Viewer

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

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 FISH Fluorescence Imaging Kit	491	516	75000¹	0.9¹	0.21	0.11
ReadiLink™ iFluor® 555 FISH Fluorescence Imaging Kit	557	570	100000¹	0.64¹	0.23	0.14

Images

Figure 1. Telomere quantitative fluorescence in situ hybridization in metaphase HeLa cells using iFluor® 647-dUTP labeled telomere probes. Probes were created using the ReadiLink™ iFluor® 647 FISH Fluorescence Imaging kit.

References

View all 32 references: Citation Explorer

Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immunostaining.
Authors: Paramasivam, Prasath and Stöter, Martin and Corradi, Eloina and Dalla Costa, Irene and Höijer, Andreas and Bartesaghi, Stefano and Sabirsh, Alan and Lindfors, Lennart and Yanez Arteta, Marianna and Nordberg, Peter and Andersson, Shalini and Baudet, Marie-Laure and Bickle, Marc and Zerial, Marino
Journal: RNA (New York, N.Y.) (2022): 433-446

In Situ Super-Resolution Imaging of Telomeres with DNA-PAINT.
Authors: Liu, Yuanyuan and Ye, Xiangyu and Wang, Zhuyuan and Zong, Shenfei and Cui, Yiping
Journal: ACS omega (2022): 40512-40519

A Janus 3D DNA nanomachine for simultaneous and sensitive fluorescence detection and imaging of dual microRNAs in cancer cells.
Authors: Yang, Zezhou and Peng, Xin and Yang, Peng and Zhuo, Ying and Chai, Ya-Qin and Liang, Wenbin and Yuan, Ruo
Journal: Chemical science (2020): 8482-8488

Dual-probe RNA FRET-FISH in Yeast.
Authors: Wadsworth, Gable M and Parikh, Rasesh Y and Kim, Harold D
Journal: Bio-protocol (2018): e2867

A molecular pathology method for sequential fluorescence in situ hybridization for multi-gene analysis at the single-cell level.
Authors: Hu, Linping and Yin, Xiuxiu and Sun, Jiangman and Zetterberg, Anders and Miao, Weimin and Cheng, Tao
Journal: Oncotarget (2017): 50534-50541

mRNA detection in budding yeast with single fluorophores.
Authors: Wadsworth, Gable M and Parikh, Rasesh Y and Choy, John S and Kim, Harold D
Journal: Nucleic acids research (2017): e141

Chondroitin sulfate-polyethylenimine copolymer-coated superparamagnetic iron oxide nanoparticles as an efficient magneto-gene carrier for microRNA-encoding plasmid DNA delivery.
Authors: Lo, Yu-Lun and Chou, Han-Lin and Liao, Zi-Xian and Huang, Shih-Jer and Ke, Jyun-Han and Liu, Yu-Sheng and Chiu, Chien-Chih and Wang, Li-Fang
Journal: Nanoscale (2015): 8554-65

Quantitation and localization of pospiviroids in aphids.
Authors: Van Bogaert, N and De Jonghe, K and Van Damme, E J M and Maes, M and Smagghe, G
Journal: Journal of virological methods (2015): 51-4

CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.
Authors: Deng, Wulan and Shi, Xinghua and Tjian, Robert and Lionnet, Timothée and Singer, Robert H
Journal: Proceedings of the National Academy of Sciences of the United States of America (2015): 11870-5

Comparison of the sequence-dependent fluorescence of the cyanine dyes Cy3, Cy5, DyLight DY547 and DyLight DY647 on single-stranded DNA.
Authors: Kretschy, Nicole and Somoza, Mark M
Journal: PloS one (2014): e85605

Documents

Safety data sheet
Product protocol
Certificate of analysis

Application notes

A Novel Fluorescent Probe for Imaging and Detecting Hydroxyl Radical in Living Cells
Abbreviation of Common Chemical Compounds Related to Peptides
Annexin V
Bright Tide Fluor™-Based Fluorescent Peptides and Their Applications In Drug Discovery and Disease Diagnosis
Calcein

FAQ

Are Cell Navigator® Cell Plasma Membrane Staining Kits suitable for cell culture medium samples?
Are there any alternatives for ethidium bromide in agarose gels?
Are there any alternatives to Cy5?
Are there safer alternatives to ethidium bromide?
Can 7-AAD be fixed?

AssayWise

Nucleic Acid Quantification
Hydroxyl Radical Detection
Nucleic Acid Detection, Quantification and Imaging
A practical guide for use of PE and APC in flow cytometry
A Comprehensive Suite of Online Tools

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Parameter	Polymerase Activation	PCR (30-40 cycles)
	Hold	Denature	Anneal	Extend
Temperature	95 °C	95 °C	55-65 °C	68-72 °C
Time (m:ss)	0:20	0:30	1:00	1:00

ReadiLink™ iFluor® 647 FISH Fluorescence Imaging Kit

OverviewSDSProtocol

Platform

Thermal Cycler

Components

Example protocol

SAMPLE EXPERIMENTAL PROTOCOL

Spectrum

Spectral properties

Product Family

Images

References

Documents

Application notes

FAQ

AssayWise

Overview SDS Protocol