ReadiLink™ iFluor® 555 FISH Fluorescence Imaging Kit

Telomere quantitative fluorescence in situ hybridization in metaphase HeLa cells using iFluor® 555-dUTP labeled telomere probes. Probes were created using the ReadiLink™ iFluor® 555 FISH Fluorescence Imaging kit.

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Physical properties

Solvent

Water

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

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)

0.23

Correction Factor (280 nm)

0.14

Extinction coefficient (cm ^-1 M ^-1)

100000¹

Excitation (nm)

557

Emission (nm)

570

Quantum yield

0.64¹

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® 555 FISH fluorescence imaging kit is a convenient tool for labeling a target DNA using an iFluor® 555 labeled FISH probe via in situ hybridization. The kit provides Taq DNA polymerase enzyme which incorporates iFluor® 555-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® 555-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)	0.23
Correction Factor (280 nm)	0.14
Extinction coefficient (cm ^-1 M ^-1)	100000¹
Excitation (nm)	557
Emission (nm)	570
Quantum yield	0.64¹

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 FISH Fluorescence Imaging Kit	491	516	75000¹	0.9¹	0.21	0.11	-
ReadiLink™ iFluor® 647 FISH Fluorescence Imaging Kit	656	670	250000¹	0.25¹	0.03	0.03	0.0793

Images

Figure 1. Telomere quantitative fluorescence in situ hybridization in metaphase HeLa cells using iFluor® 555-dUTP labeled telomere probes. Probes were created using the ReadiLink™ iFluor® 555 FISH Fluorescence Imaging kit.

References

View all 49 references: Citation Explorer

Evaluation of a fluorescence in situ hybridization (FISH)-based method for detection of SARS-CoV-2 in saliva.
Authors: Tamminga, Gerrit G and Jansen, Gijsbert J and Wiersma, Marit
Journal: PloS one (2022): e0277367

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

Detection of hydrocarbon-degrading bacteria on deepwater corals of the northeast Atlantic using CARD-FISH.
Authors: Thompson, Haydn Frank and Gutierrez, Tony
Journal: Journal of microbiological methods (2021): 106277

Ammonium and organic carbon co-removal under feammox-coupled-with-heterotrophy condition as an efficient approach for nitrogen treatment.
Authors: Le, Chung Phuong and Nguyen, Hai Thi and Nguyen, Toi Duy and Nguyen, Quyen Huynh Minh and Pham, Hai The and Dinh, Hang Thuy
Journal: Scientific reports (2021): 784

Development of a Fluorescence in Situ Hybridization Probe for Detecting IKZF1 Deletion Mutations in Patients with Acute Lymphoblastic Leukemia.
Authors: Hashiguchi, Junichi and Onozawa, Masahiro and Oguri, Satoshi and Fujisawa, Shinichi and Tsuji, Masahisa and Okada, Kohei and Nakagawa, Masao and Hashimoto, Daigo and Kahata, Kaoru and Kondo, Takeshi and Shimizu, Chikara and Teshima, Takanori
Journal: The Journal of molecular diagnostics : JMD (2018): 446-454

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

Evaluation of telomere length in human cardiac tissues using cardiac quantitative FISH.
Authors: Sharifi-Sanjani, Maryam and Meeker, Alan K and Mourkioti, Foteini
Journal: Nature protocols (2017): 1855-1870

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

A stem-less probe using spontaneous pairing between Cy3 and quencher for RNA detection.
Authors: Kashida, Hiromu and Morimoto, Kazuhiro and Asanuma, Hiroyuki
Journal: Science and technology of advanced materials (2016): 267-273

Fluorescence In Vivo Hybridization (FIVH) for Detection of Helicobacter pylori Infection in a C57BL/6 Mouse Model.
Authors: Fontenete, Sílvia and Leite, Marina and Cappoen, Davie and Santos, Rita and Ginneken, Chris Van and Figueiredo, Céu and Wengel, Jesper and Cos, Paul and Azevedo, Nuno Filipe
Journal: PloS one (2016): e0148353

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

Glycerol 3-Phosphate Assays
Nucleic Acid Quantification
Hydroxyl Radical Detection
Thiol Detection
Nucleic Acid Detection, Quantification and Imaging

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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® 555 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