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Cy5 dutp

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Cy5-dUTP is a fluorescently labeled nucleotide used in various molecular biology and genomics applications. It can be incorporated into DNA or RNA molecules for detection and analysis purposes.

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Lab products found in correlation

Cy3 dutp, by PerkinElmer (5 mentions) Fluorescein dutp, by Enzo Life Sciences (2 mentions) Microcon centrifugal filter device, by Merck Group (1 mentions) Agilent microarray scanner, by Agilent Technologies (1 mentions) Agilent feature extraction software v 8, by Agilent Technologies (1 mentions) Agilent cgh analytics software v3.4, by Agilent Technologies (1 mentions) Whole genome amplification kit, by Merck Group (1 mentions)

5 protocols using cy5 dutp

1

Primate Cytogenetic Analysis by FISH

Cited 1 time
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Interphase nuclei and metaphase spreads were obtained from lymphoblast and fibroblast cell lines from 20 human HapMap individuals (Coriell Cell Repository, Camden, NJ), four chimpanzees (Katie; Veronica; Cochise; PTR8), two gorillas (GGO5; GGO8) and three orangutans (PPY9; PPY16; PPY13). All cell lines were tested for mycoplasma contamination. Primate cell lines were previously collected at the University of Washington and at the University of Bari (Supplementary Table 17) and have not been authenticated. FISH experiments were performed using fosmid clones directly labeled by nick-translation with Cy3-dUTP (PerkinElmer), Cy5-dUTP (PerkinElmer), and fluorescein-dUTP (Enzo) as described previously23 (link). A minimum of 50 interphase cells were scored for each inversion to statistically determine the orientation of the examined region.
Antonacci F., Dennis M.Y., Huddleston J., Sudmant P.H., Steinberg K.M., Rosenfeld J.A., Miroballo M., Graves T.A., Vives L., Malig M., Denman L., Raja A., Stuart A., Tang J., Munson B., Shaffer L.G., Amemiya C.T., Wilson R.K, & Eichler E.E. (2014). Palindromic GOLGA8 core duplicons promote chromosome 15q13.3 microdeletion and evolutionary instability. Nature genetics, 46(12), 1293-1302.
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2

Genome-wide Array CGH Analysis

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Array CGH was performed using a human genome-wide 185K oligonucleotide array (Agilent Technologies). Genomic DNA from the inversion patient (II-4, family #1) and from a sex-matched reference were double-digested separately using the restriction endonucleases AluI and RsaI (Promega) and purified using Microcon centrifugal filter devices (Merck Millipore). A total of 1.5 μg of the digested products was differentially labelled by the random priming method using the fluorophores Cy3-dUTP and Cy5-dUTP (Perkin Elmer) and co-hybridised to the array for 48 h at 65°C in a rotating oven. The hybridised arrays were washed and scanned using an Agilent Microarray Scanner. The image data were extracted using Agilent Feature Extraction software V.8.5, and the data analysed using Agilent CGH Analytics software V.3.4 (z-score method setting).
Babbs C., Lloyd D., Pagnamenta A.T., Twigg S.R., Green J., McGowan S.J., Mirza G., Naples R., Sharma V.P., Volpi E.V., Buckle V.J., Wall S.A., Knight S.J., Parr J.R, & Wilkie A.O. (2014). De novo and rare inherited mutations implicate the transcriptional coregulator TCF20/SPBP in autism spectrum disorder. Journal of Medical Genetics, 51(11), 737-747.
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3

Methylated DNA Immunoprecipitation for Microarray

Cited 1 time
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The MeDIP analysis was adapted from Keshet et al. (2006) (link) and described in Provencal et al. (2012) (link)). Briefly, 2 μg PFC DNA of the eight monkeys was sonicated and methylated DNA was immunoprecipitated with 10 μg of anti-5methylcytosine (EMD Millipore, Billerica, MA,USA). The input and bound fraction were then amplified in triplicate using the Whole Genome Amplification kit (Sigma-Aldrich, Oakville, ON, Canada). The amplified input and bound fractions were labeled for microarray hybridization with either Cy3-dUTP or Cy5-dUTP (Perkin Elmer, Woodbridge, ON, Canada) respectively using the CGH labeling kit (Life Technologies, Burlington, ON, Canada) following the manufacturer’s instructions.
MASSART R., SUDERMAN M., PROVENCAL N., YI C., BENNETT A.J., SUOMI S, & SZYF M. (2014). HYDROXYMETHYLATION AND DNA METHYLATION PROFILES IN THE PREFRONTAL CORTEX OF THE NON-HUMAN PRIMATE RHESUS MACAQUE AND THE IMPACT OF MATERNAL DEPRIVATION ON HYDROXYMETHYLATION. Neuroscience, 268, 139-148.
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4

Primate Cytogenetic Analysis by FISH

Cited 1 time
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Interphase nuclei and metaphase spreads were obtained from lymphoblast and fibroblast cell lines from 20 human HapMap individuals (Coriell Cell Repository, Camden, NJ), four chimpanzees (Katie; Veronica; Cochise; PTR8), two gorillas (GGO5; GGO8) and three orangutans (PPY9; PPY16; PPY13). All cell lines were tested for mycoplasma contamination. Primate cell lines were previously collected at the University of Washington and at the University of Bari (Supplementary Table 17) and have not been authenticated. FISH experiments were performed using fosmid clones directly labeled by nick-translation with Cy3-dUTP (PerkinElmer), Cy5-dUTP (PerkinElmer), and fluorescein-dUTP (Enzo) as described previously23 (link). A minimum of 50 interphase cells were scored for each inversion to statistically determine the orientation of the examined region.
Antonacci F., Dennis M.Y., Huddleston J., Sudmant P.H., Steinberg K.M., Rosenfeld J.A., Miroballo M., Graves T.A., Vives L., Malig M., Denman L., Raja A., Stuart A., Tang J., Munson B., Shaffer L.G., Amemiya C.T., Wilson R.K, & Eichler E.E. (2014). Palindromic GOLGA8 core duplicons promote chromosome 15q13.3 microdeletion and evolutionary instability. Nature genetics, 46(12), 1293-1302.
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5

Fluorescent In Situ Hybridization Protocol

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The array-CGH results were con rmed by uorescence in situ hybridization (FISH). Metaphase spreads of each investigated individual (42/19, 43/19, 129/20, 145/20) were obtained from phytohaemagglutinin (PHA)-stimulated whole blood cultures. FISH experiments were performed using human BAC clones (table 1) directly labeled by nick-translation with Cy3-dUTP (Perkin-Elmer), Cy5-dUTP (Perkin-Elmer) and uorescein-dUTP (Enzo) as described by Lichter et al. (Lichter et al. 1990), with minor modi cations. Brie y, 300 ng of labeled probe were used for the FISH experiments; hybridization was performed at 37oC in 2xSSC, 50% (v/v) formamide, 10% (w/v) dextran sulphate and 3 μg sonicated salmon sperm DNA, in a volume of 10 μL. Posthybridization washing was at high stringency (60°C in 0.1xSSC, three times). Fluorescence signal intensity from DAPI, Cy3, Cy5 and uorescein was detected with speci c lters using a Leica DMRXA2 epi uorescence microscope (Leica Microsystems GmbH, Wetzlar, Hesse, Germany) equipped with a cooled CCD camera. Digital images were separately recorded as grayscale pictures and subsequently pseudocolored and merged using Adobe Photoshop software (Adobe, San Jose, California, United States).
Dell’Edera D., Allegretti A., Rosalba A.D., Dell'Edera M.T., Epifania A.A., Mercuri L., Mitidieri A., Simone F., & Ventura M. (2020). 7q35q36.3 Deletion and Concomitant 20q13.2q13.33 Duplication in a Newborn: Familiar Case.
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