HeLa cells cultured on coverslips were washed in PBS prior to fixation in 4% para-formaldehyde, 1× PBS for 10 min at RT. Cells were permeabilized 6 min with 0.2% Triton-X-100 in PBS then pre-equilibrated in 2× SCC and treated with RNase A (100 µg/mL) at 37 °C, 1 h. Following washing in 2× SCC, cells on coverslips were dehydrated with a 70%, 85%, 100% ethanol series, then air dried, heated to 70 °C and submerged into 85 °C preheated 70% formamide, 2× SSC (pH 7.0) for 18 min. A second ethanol dehydration series was performed. 150–300 ng biotin-labelled probe was added in hybridization buffer (50% formamide, 2× SSC, 1% Tween20, 10% Dextran Sulphate) containing 6 µg human Cot1 DNA (Invitrogen, Waltham, MA, USA) and sheared salmon sperm DNA and incubated at 37 °C, 24 h. An HSV-1 ICP27 plasmid was end-labelled. After incubation with this probe, coverslips were washed 4 × 5 min in 4× SSC, 50 °C, followed by 4 × 5 min washes in 0.1× SSC, 65 °C, then pre-equilibrated in 4× SSC, 0.1% Tween-20 and blocked with 4% BSA before incubating 30 min, RT with Alexa Fluor® (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) conjugated-Steptavidin antibodies and 4,6-diamidino-2 phenylindole, dihydrochloride (DAPI) at 2 µg/mL. Coverslips were washed 3 times in 4× SSC, 0.1% Tween-20, 37 °C and mounted on slides in Vectashield (Vector Labs).
Human cot 1 dna
Manufactured by Thermo Fisher Scientific
Sourced in United States, Switzerland
Human Cot-1 DNA is a nucleic acid product designed for use in molecular biology and genetic research applications. It is derived from the repetitive sequences of the human genome known as Cot-1 DNA. This product can be used as a tool for various techniques, such as Northern blotting, Southern blotting, and hybridization analysis.
Automatically generated - may contain errors
Lab products found in correlation
Xgen universal blocking oligos, by Integrated DNA Technologies (7 mentions)
Kapa hyper prep kit, by Roche (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Digoxigenin labeled, by Roche (4 mentions)
Agencourt ampure xp bead, by Beckman Coulter (4 mentions)
Hiseq 4000 ngs platforms, by Illumina (4 mentions)
Dynabeads m 270, by Thermo Fisher Scientific (4 mentions)
Nimblegen seqcap ez hybridization and wash kit, by Roche (4 mentions)
Cellcarrier plate, by PerkinElmer (3 mentions)
Salmon sperm dna, by Thermo Fisher Scientific (3 mentions)
Ampure xp beads, by Roche (3 mentions)
Kapa library quantification kit, by Roche (3 mentions)
Kapa hifi hotstart readymix, by Roche (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Janus liquid handler workstation, by PerkinElmer (2 mentions)
Dna blocker, by Integrated DNA Technologies (2 mentions)
Xgen lockdown probes kit, by Integrated DNA Technologies (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Axioimager z2 fluorescence microscope, by Zeiss (2 mentions)
49 protocols using human cot 1 dna
1
Fluorescence In Situ Hybridization Assay for HSV-1 ICP27 in HeLa Cells
2
High-Throughput FISH Assay for Hypoxic Cells
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For high-throughput FISH, cells were plated in 384-well CellCarrier plates (PerkinElmer) at a concentration of 2000 cells/well. After normoxic or hypoxic culture for up to 48 h, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min. After two washes with PBS, cells were permeabilized in 0.5% Triton X-100, 0.5% Saponin/PBS for 20 min at room temperature (RT) and then incubated in 0.1 N HCl for 15 min at RT. Cells were kept in 50% formamide/2× SSC for at least 30 min at RT. A probe mix containing 60 ng of each library probe and fluorescently labeled readout probe and 40 μg human COT1 DNA (Invitrogen) in 1.1 ml of hybridization buffer (20% dextran sulfate, 50% formamide, 2× SSC, 1× Denhartd’s solution) was used; 15 µl of probe mix was added to the corresponding wells of a 384-well plate prepared using a JANUS liquid handler workstation (PerkinElmer, Waltham, MA). Probes were denatured at 85°C for 7 min, and the plate was incubated at 37°C overnight (16 h) for hybridization. After incubation, excess probe was washed off 3 times with 100 µl 2× SSC, 2× SSC at 42°C, and 2× SSC at 60°C for 5 min each. Cells were stained with DAPI in PBS (5 ng/ml) before imaging.
3
Enrichment and Sequencing of Viral DNA
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For the enrichment of viral fragments contained in the synthesized libraries, several libraries were pooled to perform the capture step, as described before31 (link). Briefly, the pooled libraries were mixed with the virus-specific probes in the presence of human Cot-1 DNA (Invitrogen) and xGen universal blocking oligos (IDT) for the hybridization step. A series of wash steps were performed using DNA xGen lockdown reagents (IDT), following the manufacturer’s recommendations. The quality of the enriched DNA libraries was evaluated by electrophoresis with a TapeStation 2200 system (Agilent Technologies) and quantified by real time PCR with the GenNext NGS library quantification kit (Toyobo). Finally, the multiplexed libraries were subjected to cluster generation using a MiSeq Reagent Kit v3 (150 cycles) or NextSeq 500 Kit (75 cycles) in MiSeq or NextSeq desktop sequencing systems (Illumina), respectively.
4
Mapping X-Chromosome Inactivation by FISH
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For XIST RNA FISH, a combination of two probes covering 16 kb of XIST mRNA was used (Okamoto et al. 2011 (link)). For nascent transcript detection by RNA FISH, the following BAC (CHORI) probes were used: HDAC8 (RP11-1021B19), TBL1X (RP11-451G24), ATRX (RP11-42M11), HUWE1 (RP11-155O24), and KDM5C (RP11-258C19). The correct chromosomal location of BACs was first verified using DNA FISH on metaphase spreads. A FISH probe for MAGEA6 was generated by cloning the genomic sequence in pCR-XL-TOPO vector. Human Cot-1 DNA (Invitrogen) was used for Cot-1 RNA FISH. Probes were labeled by nick translation (Vysis) with Spectrum Red-dUTP, Spectrum Green-dUTP, or Cy5-dUTP following the manufacturer's instructions. RNA and DNA FISH were performed as described previously (Chaumeil et al. 2008 (link)). For more details see Supplemental Methods.
5
Chromosomal Mapping of Rat ESCs and Brain
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The trypsinized rat ESCs and homogenized brain samples were incubated for 15 min in 0.075 M KCl, fixed with methanol and acetic acid (3:1), and then slides were prepared using standard methods. FISH analyses were performed using fixed metaphase spreads of each cell hybrid using digoxigenin-labeled (Roche, Basel, Switzerland) DNA [mouse COT-1 DNA] and biotin-labeled DNA [human COT-1 DNA (Invitrogen)]51 (link). Chromosomal DNA was counterstained with DAPI (Sigma-Aldrich). Images were captured using an AxioImagerZ2 fluorescence microscope (Carl Zeiss GmbH).
6
Genomic DNA Analysis via Array CGH
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Genomic DNA of each cell line was analyzed using Array CGH with the latest version of OncoBAC arrays for DNA copy. The OncoBAC arrays were comprised of 2402 P1, PAC, or BAC clones39 (link). About 80% of the clones on the OncoBAC arrays contained genes and STSs implicated in cancer development or progression. All clones were printed in quadruplicate. DNA samples for array CGH were labeled generally as described previously40 (link)–42 (link). Briefly, we random-prime labeled 500 ng of test (cell line) and reference (normal female, Promega) genomic DNA with CY3-dUTP and CY5-dUTP (GE Healthcare Life Science), respectively, using Bioprime kit (Invitrogen). Labeled DNA samples were coprecipitated with 50 mg of human Cot-1 DNA (Invitrogen), denatured, hybridized to BAC arrays for 48–72 h, washed, and counterstained with DAPI. Data processing Array CGH data image analyses were performed as described previously43 (link). Array probes missing in more than 50% of samples in the OncoBAC or scanning array data sets were excluded in the corresponding set. Array probes representing the same DNA sequence were averaged within each data set.
7
Enrichment of Proviral DNA Sequences
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After library synthesis, up to 6 samples, containing different Index sequences, were mixed together in order to reach the recommended amount of 500 ng, to perform the enrichment step involving hybridization with the virus-specific probes. This was done using the SeqCap EZ Hybridization and Wash Kit (Roche NimbleGen) following instructions of the rapid protocol for DNA probe hybridization and target capture from IDT. Briefly, library DNA was first mixed with 5 μg of human Cot-1 DNA (Invitrogen) and xGen Universal Blocking Oligos (IDT) and dried up using an evaporator (Eyela). The dried DNA was the dissolved in the hybridization buffer. After an incubation step of 10 minutes at 95 °C, the probes were added and hybridization was allowed to take place at 65 °C for 4h. Streptavidin-coated magnetic beads (Life Technologies) were added to the hybridization mixture, and the sample was additionally incubated for 45 minutes at 65 °C. After the recommended washing steps, the captured DNA was amplified by PCR and further purified using Agencourt AMPure XP beads (Beckman Coulter). DNA libraries enriched for proviral sequences were quantified by qPCR using Illumina’s P5 and P7 primers prior to the sequencing step.
8
BAC Probes Preparation and Labeling
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All BAC clones were ordered from the BACPAC Resource Center at the Children’s Hospital Oakland Research Institute: “U” probe is RP11-74P5, “C” probe is RP11-337 N12, and “D” probe is RP11-248 M23. BAC DNAs were labeled with either Chromatide Alexa Fluor 488-5 dUTP (Invitrogen, C-11397) or Alexa Fluor 647-aha-dUTP (Invitrogen, A32764) using nick-translation kit (Roche, 10976776001), and incubated in 15 °C for 4 h. The nick-translation reaction was deactivated using 1 μL of 0.5 M EDTA, pH 8.0, and heated for 10 min at 65 °C. The probes were then purified using illustra ProbeQuant G-50 Micro Columns (GE Healthcare, 28903408) and eluted to a concentration of 20 ng/μL. Probes were mixed with Human Cot-1 DNA (Invitrogen, 15279011) and salmon sperm (Invitrogen, 15632011), and precipitated with 1/10th volume of 3 M sodium acetate, pH 5.2, and 2.5 volume of absolute ethanol for at least 2 h at − 20 °C. Probes were then spun down, washed with cold 70% ethanol, resuspended in formamide and 40% dextran sulfate in 8X SSC, and incubated at 55 °C.
9
Fluorescence in situ Hybridization for Detecting HPV16
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Cells grown on coverslips were fixed in cold methanol-acetic acid (3:1) for 1–3 minutes followed by 4% paraformaldehyde–PBS for 10 minutes. Cells were treated with RNase A for 1 hour at 37°C and dehydrated in a 70%, 90%, and 100% ethanol series for 3 minutes each. Green 5-Fluorescein dUTP labelled BAC clone RP11-1140H22 (chr2: 28,504,596–28,660,966; hg19) was purchased from Empire Genomics. DNA-FISH probe against full-length HPV16 DNA was fluorescently labeled using the Alexa Fluor-594 FISH Tag DNA Multicolor Kit (Life Technologies) following manufacturer’s protocol. To each coverslip, 40–50 ng labeled DNA FISH-probe in hybridization buffer (Empire Genomics) supplemented with 50 μg/ml human Cot-1 DNA (Invitrogen) was added. DNA denaturation was performed at 75°C for 5 minutes, followed by hybridization at 37°C overnight. Cells were washed for 5 minutes each with 1x phosphate-buffered detergent (Qbiogene) at room temperature, 1x wash buffer (0.5x SSC, 0.1% SDS) at 65°C, and 1x phosphate-buffered detergent at room temperature. Coverslips were mounted in ProLong Gold (ThermoFisher) containing DAPI for analysis by confocal microscopy.
10
Fluorescent RNA-FISH Probe Synthesis
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RNA-FISH probes for the ∼12-kbp intergenic sequence (IGS) upstream of the rDNA start site was prepared by nick translation of the pUC9-rDNA vector (a gift from Brian McStay) with Spectrum Red-conjugated dUTP, using DNase I (5 mU/μl)-DNA polymerase I (50 mU/μl) (Roche) for 3 h at 15°C. Six micrograms of nick-translated probe was precipitated overnight at −20°C with 20 μg human Cot1 DNA (Invitrogen) and 40 μg salmon sperm DNA (Invitrogen) using cold 100% ethanol and 1/10 volume of 3 M sodium acetate. The probe was resuspended in deionized formamide at 37°C. Prior to hybridization, the probe was denatured at 80°C for 5 min, followed by addition of an equal volume of 2× hybridization mix containing 2 mM vanadyl ribonucleoside and incubation on ice for 30 min.
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