CGH analysis was performed using our custom Agilent microarray (4×44K format)12 (link). Genomic DNA from wild type or mutants was digested with AluI and RsaI. After complete digestion, mutant DNA was labeled with Cy-5 dCTP (Amersham Biosciences) and wild type DNA was labeled with Cy-3 dCTP (Amersham Biosciences) using the BioPrime® Array CGH Genomic Labeling kit (Invitrogen). Equal amounts of labeled DNA (1.5 ug) were competitively hybridized onto the microarray. Prehybridization, probe hybridization, washing, and drying steps for arrays were performed as for ChIP-chip experiments12 (link). Arrays were scanned using an Agilent scanner (Agilent) and analyzed using Agilent Feature Extraction (Agilent). Signal intensity ratios between Cy5 (mutant) and Cy3 (Wild type) were calculated from rProcessedSignal and gProcessedSignal values according to Agilent Feature Extraction. The log2 transformed Cy5/Cy3 ratio is plotted along the chromosome.
Cy3 dctp
Manufactured by Cytiva
Sourced in United Kingdom, United States
Cy3-dCTP is a fluorescently-labeled nucleotide used for labeling DNA or RNA in molecular biology applications. It contains the cyanine 3 (Cy3) dye molecule attached to the 2'-deoxycytidine-5'-triphosphate (dCTP) nucleotide.
Automatically generated - may contain errors
Lab products found in correlation
Cy5 dctp, by Cytiva (3 mentions)
Agilent scanner, by Agilent Technologies (1 mentions)
Bioprime array cgh genomic labeling kit, by Thermo Fisher Scientific (1 mentions)
Agilent feature extraction, by Agilent Technologies (1 mentions)
Agilent g2565aa microarray scanner, by Agilent Technologies (1 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Rneasy lipid tissue mini kit, by Qiagen (1 mentions)
Minelute purification columns, by Qiagen (1 mentions)
Yeast trna, by Thermo Fisher Scientific (1 mentions)
Axioskop 2, by Zeiss (1 mentions)
Isis imaging software, by MetaSystems (1 mentions)
Green dutp, by Abbott (1 mentions)
Axio imager z2, by Zeiss (1 mentions)
5 protocols using cy3 dctp
1
Comparative Genomic Hybridization Analysis
2
Visualizing DNA Synthesis and Repair
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DNA polymerase mediated incorporation of thymidine analogs, 5-bromo-2’-deoxyuridine (BrdU) or 5-ethynyl-2’-deoxyuridine (EdU), was taken as a proxy for the processive DNA synthesis in DNA replication and repair experiments.
To visualize BrdU or EdU incorporation after DNA repair, medium containing 10 micromolar concentration of BrdU or EdU was added to the cells directly before microirradiation. One hour post irradiation, cells were fixed for 10 minutes with 3.7% formaldehyde in phosphate buffered saline solution (PBS) and stained for either BrdU or EdU as described below.
In DNA replication experiments to label nascent DNA in live cells, C2C12 cells expressing GFP-PCNA were scratch loaded [69 ] with Cy3-dCTP (20 micromolar, Amersham) in complete culture medium and incubated for 20 minutes before replacing the medium. Cells were cultured overnight before performing live cell microscopy.
In double pulse nucleotide labeling experiments, C2C12 cells expressing fluorescent PCNA were first scratch loaded with either Cy3-dCTP or Atto488-dCTP, and incubated in the culture medium for the indicated period of time. EdU at the final concentration of 10 micromolar was then added to the medium and cells were cultured further until fixation with 3.7% formaldehyde in PBS.
To visualize BrdU or EdU incorporation after DNA repair, medium containing 10 micromolar concentration of BrdU or EdU was added to the cells directly before microirradiation. One hour post irradiation, cells were fixed for 10 minutes with 3.7% formaldehyde in phosphate buffered saline solution (PBS) and stained for either BrdU or EdU as described below.
In DNA replication experiments to label nascent DNA in live cells, C2C12 cells expressing GFP-PCNA were scratch loaded [
In double pulse nucleotide labeling experiments, C2C12 cells expressing fluorescent PCNA were first scratch loaded with either Cy3-dCTP or Atto488-dCTP, and incubated in the culture medium for the indicated period of time. EdU at the final concentration of 10 micromolar was then added to the medium and cells were cultured further until fixation with 3.7% formaldehyde in PBS.
3
Transcriptomic Analysis of Mouse Tissues
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Oligo nucleotide microarrays were produced at the SCIBLU Genomics Center, Lund University, Sweden (www.lth.se/sciblu ) using a set of ~ 37,000 mouse oligonucleotide probes (Operon Ver. 4.0) as previously described [14 (link)]. Total RNA was extracted from 10 to 15 mg frozen tissue, using RNeasy lipid tissue mini kit (Qiagen, Valencia, CA, USA) and quality controlled using a BioAnalyzer 2100 system (Agilent Technologies, Kista Sweden). The Universal Mouse reference RNA (Stratagene, La Jolla, CA, USA) was used as reference in all experiments. Fluorescently labeled cDNA targets for hybridization were prepared according to manufacturers’ instructions using the Corning Pronto Plus System 6 (Corning Life Sciences, NY, USA). Samples were labeled with Cy3-dCTP (Amersham) and reference was labeled with Cy5-dCTP. Hybridization was performed overnight at 42 °C using a MAUI hybridization system (BioMicro Systems, Salt Lake City, USA), and slides were washed according to the Corning Pronto Plus system instructions. Fluorescence was recorded using an Agilent G2565AA microarray scanner (Agilent Technologies).
4
Breast Cancer Cell Line DNA Analysis
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Cell line DNA was analysed on the Breakthrough Breast Cancer human CGH 4.6K 1.12 arrays as previously described
[13 (link)]. Briefly, 1 μg of test and normal female genomic DNA, from pooled donor samples, was directly labelled with Cy3-dCTP or Cy5-dCTP (Amersham BioSciences, Amersham, UK) using a Bioprime labelling kit (Invitrogen, Paisley, UK) according to the manufacturer's protocol modified to incorporate 1.0 mM Cy dye, 0.6 mM dCTP, and 1.2 mM dATP, dGTP and dTTP. Unincorporated nucleotides were removed with MinElute purification columns (Qiagen, Crawley, UK). The labelled DNA was co-precipitated with 100 μg of Cot1 (Invitrogen, Paisley, UK), resuspended in hybridization buffer [50% formamide, 10% dextran sulphate, 2× SSC, 2% SDS, 2 mg of yeast tRNA (Invitrogen, Paisley, UK)], denatured at 75°C for 5 min, and pre-annealed for 30 min at 37°C. Slides were blocked in 10% BSA–50% formamide solution at 42°C for 45 min. The probe was subsequently applied to the slide and hybridized overnight at 42°C. Slides were washed in 2× SSC, 0.1% SDS for 15 min at 45°C; 2× SSC, 50% formamide for 15 min at 45°C; 2× SSC, 0.1% SDS for a subsequent 30 min at 45°C; and finally two 15-min washes of 0.2× SSC at room temperature. Slides were centrifuged at 1200 rpm for 2 min to dry. Each experiment was performed in duplicate as a dye swap to eliminate any labelling bias.
[13 (link)]. Briefly, 1 μg of test and normal female genomic DNA, from pooled donor samples, was directly labelled with Cy3-dCTP or Cy5-dCTP (Amersham BioSciences, Amersham, UK) using a Bioprime labelling kit (Invitrogen, Paisley, UK) according to the manufacturer's protocol modified to incorporate 1.0 mM Cy dye, 0.6 mM dCTP, and 1.2 mM dATP, dGTP and dTTP. Unincorporated nucleotides were removed with MinElute purification columns (Qiagen, Crawley, UK). The labelled DNA was co-precipitated with 100 μg of Cot1 (Invitrogen, Paisley, UK), resuspended in hybridization buffer [50% formamide, 10% dextran sulphate, 2× SSC, 2% SDS, 2 mg of yeast tRNA (Invitrogen, Paisley, UK)], denatured at 75°C for 5 min, and pre-annealed for 30 min at 37°C. Slides were blocked in 10% BSA–50% formamide solution at 42°C for 45 min. The probe was subsequently applied to the slide and hybridized overnight at 42°C. Slides were washed in 2× SSC, 0.1% SDS for 15 min at 45°C; 2× SSC, 50% formamide for 15 min at 45°C; 2× SSC, 0.1% SDS for a subsequent 30 min at 45°C; and finally two 15-min washes of 0.2× SSC at room temperature. Slides were centrifuged at 1200 rpm for 2 min to dry. Each experiment was performed in duplicate as a dye swap to eliminate any labelling bias.
5
Fluorescence in situ Hybridization Analysis
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Fluorescence in situ hybridization analyses were performed on chromosome preparations generated from the cell lines using the following conventional techniques: colcemid arrest, hypotonic treatment, and methanol/glacial acetic acid fixation, as described previously [34 (link)]. The following bacterial artificial chromosome (BAC) clones were used: CH82-199H02 and CH82-179B09 for the MDM2 region, CH82-213B06 and CH82-204K11 for the CDK4 region, CH82-99P23 and CH82-60O16 for the CFA 7 region (58.4 Mb to 58.9 Mb), CH82-1E17 and CH82-40I15 for the region containing GABPBP1, and USP8 and TRPM7 amplifications (https://bacpacresources.org/ , accessed on 1 July 2021). These BAC clones were labeled using green-dUTP (Abbott Molecular, Des Plaines, IL, USA) and Cy3-dCTP (Amersham Biosciences, Chalfont, UK). The slides were analyzed by an experienced cytogeneticist (FC) using a fluorescence microscope (Axioskop2, Axio Imager Z2, Zeiss, Göttingen, Germany) and Isis imaging software (Metasystems, Altlussheim, Germany). At least 100 non-overlapping tumor nuclei were examined in this study.
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