datp  (Thermo Fisher)


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    Name:
    Biotin 14 dATP
    Description:
    Biotin 14 dATP is a patented dATP analog with biotin attached at the 6 position of the purine base by a 14 atom linker The long 14 atom spacer arm allows accessibility to streptavidin for sensitive detection of probe target hybrids in in situ and filter based hybridizations Biotin 14 dATP can be efficiently incorporated into DNA by nick translation in the presence of dCTP dGTP and dTTP 1 Biotin 14 dATP can be added to the 3 end of DNA using terminal deoxynucleotidyl transferase 2 The amount supplied is sufficient for labeling up to 50 µg of DNA by nick translation Performance and Quality Testing Purity is evaluated by reverse phase HPLC analysis
    Catalog Number:
    19524016
    Price:
    None
    Category:
    Oligos Primers Probes Nucleotides
    Applications:
    DNA & RNA Purification & Analysis|DNA Labeling|Nucleic Acid Labeling & Oligo Synthesis
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    Structured Review

    Thermo Fisher datp
    Addition of <t>5mdCTP</t> in UniNicE-seq work flow. (A) Schematic diagram showing Nt.CviPII is blocked by 5-methylcytosine in recognition (CCD) sequence. During nick translation 5mdCTP can be incorporated at one or both cytosine position. Nucleotide mixtures containing biotinylated dCTP and 5mdCTP would allow both labels at CCD sites. Lower panel shows blocking of nicking by the presence of 5mC at CCD sites. After the nicking reaction the denatured oligonucleotides were resolved in a urea-acrylamide denaturing gel by electrophoresis. C is control input oligos, − or + indicate absence or presence of Nt.CviPII in the reaction. mC and btC represents 5-methylcytosine and biotinylated-cytosine respectively. Nicked oligonucleotide migrates faster on the denaturing urea gel. (B) Schematic diagram of UniNicE-seq method for accessible chromatin library preparation. (C) Substitution of <t>dATP</t> by Texas Red conjugated dATP will allow accessible chromatin visualization in the nucleus. (D) IGV screen shot of the normalized read density of UniNicE-seq using 2.5U (top track), 25U (middle track) and 50U (bottom track) of Nt.CviPII in HCT116 (E) Venn diagram showing overlap of peaks in various amounts of Nt.CviPII in the universal NicE-seq (UniNicE-seq) reaction in HCT116 cells.
    Biotin 14 dATP is a patented dATP analog with biotin attached at the 6 position of the purine base by a 14 atom linker The long 14 atom spacer arm allows accessibility to streptavidin for sensitive detection of probe target hybrids in in situ and filter based hybridizations Biotin 14 dATP can be efficiently incorporated into DNA by nick translation in the presence of dCTP dGTP and dTTP 1 Biotin 14 dATP can be added to the 3 end of DNA using terminal deoxynucleotidyl transferase 2 The amount supplied is sufficient for labeling up to 50 µg of DNA by nick translation Performance and Quality Testing Purity is evaluated by reverse phase HPLC analysis
    https://www.bioz.com/result/datp/product/Thermo Fisher
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    datp - by Bioz Stars, 2021-05
    95/100 stars

    Images

    1) Product Images from "Universal NicE-seq for high resolution accessible chromatin profiling for formaldehyde fixed and FFPE tissues"

    Article Title: Universal NicE-seq for high resolution accessible chromatin profiling for formaldehyde fixed and FFPE tissues

    Journal: bioRxiv

    doi: 10.1101/2020.04.27.064592

    Addition of 5mdCTP in UniNicE-seq work flow. (A) Schematic diagram showing Nt.CviPII is blocked by 5-methylcytosine in recognition (CCD) sequence. During nick translation 5mdCTP can be incorporated at one or both cytosine position. Nucleotide mixtures containing biotinylated dCTP and 5mdCTP would allow both labels at CCD sites. Lower panel shows blocking of nicking by the presence of 5mC at CCD sites. After the nicking reaction the denatured oligonucleotides were resolved in a urea-acrylamide denaturing gel by electrophoresis. C is control input oligos, − or + indicate absence or presence of Nt.CviPII in the reaction. mC and btC represents 5-methylcytosine and biotinylated-cytosine respectively. Nicked oligonucleotide migrates faster on the denaturing urea gel. (B) Schematic diagram of UniNicE-seq method for accessible chromatin library preparation. (C) Substitution of dATP by Texas Red conjugated dATP will allow accessible chromatin visualization in the nucleus. (D) IGV screen shot of the normalized read density of UniNicE-seq using 2.5U (top track), 25U (middle track) and 50U (bottom track) of Nt.CviPII in HCT116 (E) Venn diagram showing overlap of peaks in various amounts of Nt.CviPII in the universal NicE-seq (UniNicE-seq) reaction in HCT116 cells.
    Figure Legend Snippet: Addition of 5mdCTP in UniNicE-seq work flow. (A) Schematic diagram showing Nt.CviPII is blocked by 5-methylcytosine in recognition (CCD) sequence. During nick translation 5mdCTP can be incorporated at one or both cytosine position. Nucleotide mixtures containing biotinylated dCTP and 5mdCTP would allow both labels at CCD sites. Lower panel shows blocking of nicking by the presence of 5mC at CCD sites. After the nicking reaction the denatured oligonucleotides were resolved in a urea-acrylamide denaturing gel by electrophoresis. C is control input oligos, − or + indicate absence or presence of Nt.CviPII in the reaction. mC and btC represents 5-methylcytosine and biotinylated-cytosine respectively. Nicked oligonucleotide migrates faster on the denaturing urea gel. (B) Schematic diagram of UniNicE-seq method for accessible chromatin library preparation. (C) Substitution of dATP by Texas Red conjugated dATP will allow accessible chromatin visualization in the nucleus. (D) IGV screen shot of the normalized read density of UniNicE-seq using 2.5U (top track), 25U (middle track) and 50U (bottom track) of Nt.CviPII in HCT116 (E) Venn diagram showing overlap of peaks in various amounts of Nt.CviPII in the universal NicE-seq (UniNicE-seq) reaction in HCT116 cells.

    Techniques Used: Sequencing, Nick Translation, Blocking Assay, Nucleic Acid Electrophoresis

    Related Articles

    Plasmid Preparation:

    Article Title: Cytogenetic characterization and genome size of the medicinal plant Catharanthus roseus (L.) G. Don
    Article Snippet: .. Plasmid DNA was labelled by nick translation using biotin-14-dATP of the Bionick™ Labeling System (Invitrogen Life Technologies, Paisley, UK). .. Plasmid DNA (0.5–1 μg) was labelled using 50 μL of labelling reaction (using 5 μL of 10× enzyme mix) for 2 h at 16 °C, and the reaction was stopped with 5 μL of 500 mM EDTA (pH 8.0).

    Nick Translation:

    Article Title: Cytogenetic characterization and genome size of the medicinal plant Catharanthus roseus (L.) G. Don
    Article Snippet: .. Plasmid DNA was labelled by nick translation using biotin-14-dATP of the Bionick™ Labeling System (Invitrogen Life Technologies, Paisley, UK). .. Plasmid DNA (0.5–1 μg) was labelled using 50 μL of labelling reaction (using 5 μL of 10× enzyme mix) for 2 h at 16 °C, and the reaction was stopped with 5 μL of 500 mM EDTA (pH 8.0).

    Article Title: Chromosome Mapping of Repetitive Sequences in Rachycentron canadum (Perciformes: Rachycentridae): Implications for Karyotypic Evolution and Perspectives for Biotechnological Uses
    Article Snippet: The second probe corresponded to a 1,400 bp segment of the 18S rRNA gene obtained via PCR from nuclear DNA (Cioffi et al. [ ]). .. The 18S rDNA probe was labeled by nick translation with DIG-11-dUTP, according to the manufacturer's specifications (Roche) and the 5S rDNA probe was labeled with biotin-14-dATP by nick translation, also according to the manufacturer's specifications (Bionick Labelling System, Invitrogen). .. The telomeric DNA sequence (TTAGGG)n was also used as a probe.

    Labeling:

    Article Title: Cytogenetic characterization and genome size of the medicinal plant Catharanthus roseus (L.) G. Don
    Article Snippet: .. Plasmid DNA was labelled by nick translation using biotin-14-dATP of the Bionick™ Labeling System (Invitrogen Life Technologies, Paisley, UK). .. Plasmid DNA (0.5–1 μg) was labelled using 50 μL of labelling reaction (using 5 μL of 10× enzyme mix) for 2 h at 16 °C, and the reaction was stopped with 5 μL of 500 mM EDTA (pH 8.0).

    Article Title: Universal NicE-seq for high resolution accessible chromatin profiling for formaldehyde fixed and FFPE tissues
    Article Snippet: The nuclei pellet was resuspended in 100 μls of 1x PBS. .. Open chromatin DNA was labeled with biotin by incubating the nuclei in presence of 2.5 U of Nt.CviPII (NEB, R0626S), 50 U of DNA polymerase I (NEB, M0209S) and 30 μM of each dGTP and dTTP, 24 μM of 5mdCTP (NEB, NO356S) and dATP 24 μM plus 6 μM of biotin-14-dATP (Invitrogen, 19524016) and 6 μM of biotin-14-dCTP (Invitrogen, 19518018) at 37°C for 2 hrs with occasional mixing. .. The labeling reaction was stopped with 20 μL of 0.5 M EDTA, and 2 μg of RNase A was added to the labeling reaction and incubated at 37°C for 0.5 hour to digest RNA.

    Article Title: The Genomic Sequences Bound to Special AT-rich Sequence-binding Protein 1 (SATB1) In Vivo in Jurkat T Cells Are Tightly Associated with the Nuclear Matrix at the Bases of the Chromatin Loops
    Article Snippet: SBS-2, SBS-3, and SBS-11 are in vivo SATB1-binding DNA sequences that were cloned by us (this study). .. For fluorescence in situ DNA hybridization (FISH), the DNA probes were labeled with biotin-14-dATP ( GIBCO BRL ) using a BioPrime DNA labeling system (Life Technologies, Gaithersburg, MD). .. The samples were incubated for 10 min in 0.1 M triethanolamine and 0.25% acetic anhydride at room temperature, denatured by incubation at 70°C for 2 min in 70% formamide, 2× SSC for 2 min, dehydrated in cold 70 and 100% ethanol for 3 min each, and allowed to air dry.

    Article Title: Chromosome Mapping of Repetitive Sequences in Rachycentron canadum (Perciformes: Rachycentridae): Implications for Karyotypic Evolution and Perspectives for Biotechnological Uses
    Article Snippet: The second probe corresponded to a 1,400 bp segment of the 18S rRNA gene obtained via PCR from nuclear DNA (Cioffi et al. [ ]). .. The 18S rDNA probe was labeled by nick translation with DIG-11-dUTP, according to the manufacturer's specifications (Roche) and the 5S rDNA probe was labeled with biotin-14-dATP by nick translation, also according to the manufacturer's specifications (Bionick Labelling System, Invitrogen). .. The telomeric DNA sequence (TTAGGG)n was also used as a probe.

    Article Title: Gene Expression Profiling in AGS Cells Stimulated with Helicobacter pylori Isogenic Strains (cagA Positive or cagA Negative)
    Article Snippet: For Northern analysis, DNase I-treated total RNA (35 μg) was separated on denaturing 1.2% agarose–formaldehyde gels and capillary transferred onto nylon membranes (Amersham Pharmacia Biotech). .. To generate the labeled probes, specific cloned cDNA fragments, amplified from the respective mRNAs (Table ), were biotinylated by PCR using Pwo polymerase (Hoffmann-La Roche, Basel, Switzerland), and 3.5 nmol of dATP was replaced by biotin-14-dATP (0.4 mM; Gibco Life Technologies, Rockville, Md.) in the reaction mixture (0.2 mM deoxynucleoside triphosphates, 2 mM MgCl2 ). .. The membranes were prehybridized for 1 h in North2South hybridization buffer (Pierce, Rockford, Ill.) at 65°C and hybridized overnight to the cDNA probe (30 ng/ml of hybridization buffer).

    Polymerase Chain Reaction:

    Article Title: A cell free system for functional centromere and kinetochore assembly Authors
    Article Snippet: Baker, catalogue # 2914) Tris-acetate (Sigma-Aldrich, catalogue # T1258) Boric acid (Sigma-Aldrich, catalogue # ) ATP (Sigma-Aldrich, catalogue # A2383) BSA (Sigma-Aldrich, catalogue # A7906) KOH (Sigma-Aldrich, catalogue # 221473) CAUTION: corrosive, causes burns, use eye and skin protection. .. QIAquick PCR purification kit (Qiagen, catalogue # 28106) Restriction enzymes EcoRI, DraI, XbaI, HaeII, AvaI (New England BioLabs, catalogue # R0101, R0129, R0145, R0107, R0152) NEB 2-log DNA ladder (New England BioLabs, catalogue # N3200) Agarose (GeneMate LE, catalogue # E-3120) Biotin-14-ATP (Invitrogen, catalogue # 19524-016) Alpha-thio-dTTP (Chemcyte, catalogue # CC-3004-1) Alpha-thio-dGTP (Chemcyte, catalogue # CC-3003-1 dCTP (Invitrogen, catalogue # 10217016) Klenow fragment (Invitrogen, catalogue # M0212) Streptavidin-FITC at 0.2mg/ml (e.g. Invitrogen catalogue # 43-4311) Polyethylene glycol (PEG) 4000 (TCI America, catalogue # P0885) HCl (J.T. .. Baker, catalogue # 9530) CAUTION: corrosive, causes burns, use eye and skin protection and a chemical fume hood.

    Article Title: Gene Expression Profiling in AGS Cells Stimulated with Helicobacter pylori Isogenic Strains (cagA Positive or cagA Negative)
    Article Snippet: For Northern analysis, DNase I-treated total RNA (35 μg) was separated on denaturing 1.2% agarose–formaldehyde gels and capillary transferred onto nylon membranes (Amersham Pharmacia Biotech). .. To generate the labeled probes, specific cloned cDNA fragments, amplified from the respective mRNAs (Table ), were biotinylated by PCR using Pwo polymerase (Hoffmann-La Roche, Basel, Switzerland), and 3.5 nmol of dATP was replaced by biotin-14-dATP (0.4 mM; Gibco Life Technologies, Rockville, Md.) in the reaction mixture (0.2 mM deoxynucleoside triphosphates, 2 mM MgCl2 ). .. The membranes were prehybridized for 1 h in North2South hybridization buffer (Pierce, Rockford, Ill.) at 65°C and hybridized overnight to the cDNA probe (30 ng/ml of hybridization buffer).

    Purification:

    Article Title: A cell free system for functional centromere and kinetochore assembly Authors
    Article Snippet: Baker, catalogue # 2914) Tris-acetate (Sigma-Aldrich, catalogue # T1258) Boric acid (Sigma-Aldrich, catalogue # ) ATP (Sigma-Aldrich, catalogue # A2383) BSA (Sigma-Aldrich, catalogue # A7906) KOH (Sigma-Aldrich, catalogue # 221473) CAUTION: corrosive, causes burns, use eye and skin protection. .. QIAquick PCR purification kit (Qiagen, catalogue # 28106) Restriction enzymes EcoRI, DraI, XbaI, HaeII, AvaI (New England BioLabs, catalogue # R0101, R0129, R0145, R0107, R0152) NEB 2-log DNA ladder (New England BioLabs, catalogue # N3200) Agarose (GeneMate LE, catalogue # E-3120) Biotin-14-ATP (Invitrogen, catalogue # 19524-016) Alpha-thio-dTTP (Chemcyte, catalogue # CC-3004-1) Alpha-thio-dGTP (Chemcyte, catalogue # CC-3003-1 dCTP (Invitrogen, catalogue # 10217016) Klenow fragment (Invitrogen, catalogue # M0212) Streptavidin-FITC at 0.2mg/ml (e.g. Invitrogen catalogue # 43-4311) Polyethylene glycol (PEG) 4000 (TCI America, catalogue # P0885) HCl (J.T. .. Baker, catalogue # 9530) CAUTION: corrosive, causes burns, use eye and skin protection and a chemical fume hood.

    Fluorescence:

    Article Title: The Genomic Sequences Bound to Special AT-rich Sequence-binding Protein 1 (SATB1) In Vivo in Jurkat T Cells Are Tightly Associated with the Nuclear Matrix at the Bases of the Chromatin Loops
    Article Snippet: SBS-2, SBS-3, and SBS-11 are in vivo SATB1-binding DNA sequences that were cloned by us (this study). .. For fluorescence in situ DNA hybridization (FISH), the DNA probes were labeled with biotin-14-dATP ( GIBCO BRL ) using a BioPrime DNA labeling system (Life Technologies, Gaithersburg, MD). .. The samples were incubated for 10 min in 0.1 M triethanolamine and 0.25% acetic anhydride at room temperature, denatured by incubation at 70°C for 2 min in 70% formamide, 2× SSC for 2 min, dehydrated in cold 70 and 100% ethanol for 3 min each, and allowed to air dry.

    In Situ:

    Article Title: The Genomic Sequences Bound to Special AT-rich Sequence-binding Protein 1 (SATB1) In Vivo in Jurkat T Cells Are Tightly Associated with the Nuclear Matrix at the Bases of the Chromatin Loops
    Article Snippet: SBS-2, SBS-3, and SBS-11 are in vivo SATB1-binding DNA sequences that were cloned by us (this study). .. For fluorescence in situ DNA hybridization (FISH), the DNA probes were labeled with biotin-14-dATP ( GIBCO BRL ) using a BioPrime DNA labeling system (Life Technologies, Gaithersburg, MD). .. The samples were incubated for 10 min in 0.1 M triethanolamine and 0.25% acetic anhydride at room temperature, denatured by incubation at 70°C for 2 min in 70% formamide, 2× SSC for 2 min, dehydrated in cold 70 and 100% ethanol for 3 min each, and allowed to air dry.

    DNA Hybridization:

    Article Title: The Genomic Sequences Bound to Special AT-rich Sequence-binding Protein 1 (SATB1) In Vivo in Jurkat T Cells Are Tightly Associated with the Nuclear Matrix at the Bases of the Chromatin Loops
    Article Snippet: SBS-2, SBS-3, and SBS-11 are in vivo SATB1-binding DNA sequences that were cloned by us (this study). .. For fluorescence in situ DNA hybridization (FISH), the DNA probes were labeled with biotin-14-dATP ( GIBCO BRL ) using a BioPrime DNA labeling system (Life Technologies, Gaithersburg, MD). .. The samples were incubated for 10 min in 0.1 M triethanolamine and 0.25% acetic anhydride at room temperature, denatured by incubation at 70°C for 2 min in 70% formamide, 2× SSC for 2 min, dehydrated in cold 70 and 100% ethanol for 3 min each, and allowed to air dry.

    Fluorescence In Situ Hybridization:

    Article Title: The Genomic Sequences Bound to Special AT-rich Sequence-binding Protein 1 (SATB1) In Vivo in Jurkat T Cells Are Tightly Associated with the Nuclear Matrix at the Bases of the Chromatin Loops
    Article Snippet: SBS-2, SBS-3, and SBS-11 are in vivo SATB1-binding DNA sequences that were cloned by us (this study). .. For fluorescence in situ DNA hybridization (FISH), the DNA probes were labeled with biotin-14-dATP ( GIBCO BRL ) using a BioPrime DNA labeling system (Life Technologies, Gaithersburg, MD). .. The samples were incubated for 10 min in 0.1 M triethanolamine and 0.25% acetic anhydride at room temperature, denatured by incubation at 70°C for 2 min in 70% formamide, 2× SSC for 2 min, dehydrated in cold 70 and 100% ethanol for 3 min each, and allowed to air dry.

    DNA Labeling:

    Article Title: The Genomic Sequences Bound to Special AT-rich Sequence-binding Protein 1 (SATB1) In Vivo in Jurkat T Cells Are Tightly Associated with the Nuclear Matrix at the Bases of the Chromatin Loops
    Article Snippet: SBS-2, SBS-3, and SBS-11 are in vivo SATB1-binding DNA sequences that were cloned by us (this study). .. For fluorescence in situ DNA hybridization (FISH), the DNA probes were labeled with biotin-14-dATP ( GIBCO BRL ) using a BioPrime DNA labeling system (Life Technologies, Gaithersburg, MD). .. The samples were incubated for 10 min in 0.1 M triethanolamine and 0.25% acetic anhydride at room temperature, denatured by incubation at 70°C for 2 min in 70% formamide, 2× SSC for 2 min, dehydrated in cold 70 and 100% ethanol for 3 min each, and allowed to air dry.

    Ligation:

    Article Title: Senescence-activated enhancer landscape orchestrates the senescence-associated secretory phenotype in murine fibroblasts
    Article Snippet: In situ Hi-C Briefly, 2 × 106 cells fixed in 1% formaldehyde were suspended in Hi-C lysis buffer, to which 100 U MobI restriction enzyme (NEB, R0147) was added for overnight chromatin digestion. .. Biotin-14-dATP (Life Technologies, 19524016) was used to fill DNA restriction ends prior to DNA proximity ligation and crosslink reversal. .. To make the biotin-labeled DNA suitable for high-throughput sequencing using the Illumina platform, the DNA was sheared to 300−500 bp in size using a Covaris LE220 sonicator (Covaris, Woburn, MA) for 135 s. Sheared DNA was end-repaired and size-selected using AMPure XP beads (Beckman Coulter, A63882) prior to the dATP-tailing step.

    Clone Assay:

    Article Title: Gene Expression Profiling in AGS Cells Stimulated with Helicobacter pylori Isogenic Strains (cagA Positive or cagA Negative)
    Article Snippet: For Northern analysis, DNase I-treated total RNA (35 μg) was separated on denaturing 1.2% agarose–formaldehyde gels and capillary transferred onto nylon membranes (Amersham Pharmacia Biotech). .. To generate the labeled probes, specific cloned cDNA fragments, amplified from the respective mRNAs (Table ), were biotinylated by PCR using Pwo polymerase (Hoffmann-La Roche, Basel, Switzerland), and 3.5 nmol of dATP was replaced by biotin-14-dATP (0.4 mM; Gibco Life Technologies, Rockville, Md.) in the reaction mixture (0.2 mM deoxynucleoside triphosphates, 2 mM MgCl2 ). .. The membranes were prehybridized for 1 h in North2South hybridization buffer (Pierce, Rockford, Ill.) at 65°C and hybridized overnight to the cDNA probe (30 ng/ml of hybridization buffer).

    Amplification:

    Article Title: Gene Expression Profiling in AGS Cells Stimulated with Helicobacter pylori Isogenic Strains (cagA Positive or cagA Negative)
    Article Snippet: For Northern analysis, DNase I-treated total RNA (35 μg) was separated on denaturing 1.2% agarose–formaldehyde gels and capillary transferred onto nylon membranes (Amersham Pharmacia Biotech). .. To generate the labeled probes, specific cloned cDNA fragments, amplified from the respective mRNAs (Table ), were biotinylated by PCR using Pwo polymerase (Hoffmann-La Roche, Basel, Switzerland), and 3.5 nmol of dATP was replaced by biotin-14-dATP (0.4 mM; Gibco Life Technologies, Rockville, Md.) in the reaction mixture (0.2 mM deoxynucleoside triphosphates, 2 mM MgCl2 ). .. The membranes were prehybridized for 1 h in North2South hybridization buffer (Pierce, Rockford, Ill.) at 65°C and hybridized overnight to the cDNA probe (30 ng/ml of hybridization buffer).

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    Thermo Fisher biotin 14 datp
    Schematic illustration of the principle of antarctic thermal-sensitive uracil-DNA-glycosylase-supplemented polymerase spiral reaction (ATSU-PSR) technique for eliminating carryover contamination. Two steps are needed for ATSU-PSR technique for removing carryover contamination. During the first stage, all PSR complicons labeled with dUTP in the presence of Bst 2.0 enzyme and dUTP. During the second stage, all subsequent ATSU-PSR amplifications are digested using ATSU, which specifically cleave carryover contaminants by removing uracil in amplicons from previous reactions. Importantly, ATSU is heat inactivated during the PSR amplification stage (63°C), and the digested contaminants are degraded, ensuring that only the target templates are amplified. In addition, three components, including fluorescein isothiocyanate (FITC)-labeled primer, <t>biotin-14-dCTP,</t> and <t>biotin-14-dATP,</t> are added into ATSU-PSR mixtures for forming the biotin- and FITC-attached duplex products.
    Biotin 14 Datp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotin 14 datp/product/Thermo Fisher
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    biotin 14 datp - by Bioz Stars, 2021-05
    95/100 stars
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    Schematic illustration of the principle of antarctic thermal-sensitive uracil-DNA-glycosylase-supplemented polymerase spiral reaction (ATSU-PSR) technique for eliminating carryover contamination. Two steps are needed for ATSU-PSR technique for removing carryover contamination. During the first stage, all PSR complicons labeled with dUTP in the presence of Bst 2.0 enzyme and dUTP. During the second stage, all subsequent ATSU-PSR amplifications are digested using ATSU, which specifically cleave carryover contaminants by removing uracil in amplicons from previous reactions. Importantly, ATSU is heat inactivated during the PSR amplification stage (63°C), and the digested contaminants are degraded, ensuring that only the target templates are amplified. In addition, three components, including fluorescein isothiocyanate (FITC)-labeled primer, biotin-14-dCTP, and biotin-14-dATP, are added into ATSU-PSR mixtures for forming the biotin- and FITC-attached duplex products.

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Simultaneous Nucleic Acids Detection and Elimination of Carryover Contamination With Nanoparticles-Based Biosensor- and Antarctic Thermal Sensitive Uracil-DNA-Glycosylase-Supplemented Polymerase Spiral Reaction

    doi: 10.3389/fbioe.2019.00401

    Figure Lengend Snippet: Schematic illustration of the principle of antarctic thermal-sensitive uracil-DNA-glycosylase-supplemented polymerase spiral reaction (ATSU-PSR) technique for eliminating carryover contamination. Two steps are needed for ATSU-PSR technique for removing carryover contamination. During the first stage, all PSR complicons labeled with dUTP in the presence of Bst 2.0 enzyme and dUTP. During the second stage, all subsequent ATSU-PSR amplifications are digested using ATSU, which specifically cleave carryover contaminants by removing uracil in amplicons from previous reactions. Importantly, ATSU is heat inactivated during the PSR amplification stage (63°C), and the digested contaminants are degraded, ensuring that only the target templates are amplified. In addition, three components, including fluorescein isothiocyanate (FITC)-labeled primer, biotin-14-dCTP, and biotin-14-dATP, are added into ATSU-PSR mixtures for forming the biotin- and FITC-attached duplex products.

    Article Snippet: Biotin-14-dCTP and biotin-14-dATP were obtained from Thermo Scientific Co., Ltd. (Shanghai, China).

    Techniques: Labeling, Amplification

    Outline of nanoparticle-based biosensor-supplemented polymerase spiral reaction assay (NB-PSR). (A) Outline of PSR with Ft* primer, biotin-14-dCTP, and biotin-14-dATP. (B) The detailed structure of NB. (C) The schematic illustration of the principle of NB for visualization of PSR products. (D) Interpretation of the results: (I) positive (two red bands, including test line and control line, appeared on the visual region of NB); (II) negative (only the control line region showed a red band).

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Simultaneous Nucleic Acids Detection and Elimination of Carryover Contamination With Nanoparticles-Based Biosensor- and Antarctic Thermal Sensitive Uracil-DNA-Glycosylase-Supplemented Polymerase Spiral Reaction

    doi: 10.3389/fbioe.2019.00401

    Figure Lengend Snippet: Outline of nanoparticle-based biosensor-supplemented polymerase spiral reaction assay (NB-PSR). (A) Outline of PSR with Ft* primer, biotin-14-dCTP, and biotin-14-dATP. (B) The detailed structure of NB. (C) The schematic illustration of the principle of NB for visualization of PSR products. (D) Interpretation of the results: (I) positive (two red bands, including test line and control line, appeared on the visual region of NB); (II) negative (only the control line region showed a red band).

    Article Snippet: Biotin-14-dCTP and biotin-14-dATP were obtained from Thermo Scientific Co., Ltd. (Shanghai, China).

    Techniques:

    Addition of 5mdCTP in UniNicE-seq work flow. (A) Schematic diagram showing Nt.CviPII is blocked by 5-methylcytosine in recognition (CCD) sequence. During nick translation 5mdCTP can be incorporated at one or both cytosine position. Nucleotide mixtures containing biotinylated dCTP and 5mdCTP would allow both labels at CCD sites. Lower panel shows blocking of nicking by the presence of 5mC at CCD sites. After the nicking reaction the denatured oligonucleotides were resolved in a urea-acrylamide denaturing gel by electrophoresis. C is control input oligos, − or + indicate absence or presence of Nt.CviPII in the reaction. mC and btC represents 5-methylcytosine and biotinylated-cytosine respectively. Nicked oligonucleotide migrates faster on the denaturing urea gel. (B) Schematic diagram of UniNicE-seq method for accessible chromatin library preparation. (C) Substitution of dATP by Texas Red conjugated dATP will allow accessible chromatin visualization in the nucleus. (D) IGV screen shot of the normalized read density of UniNicE-seq using 2.5U (top track), 25U (middle track) and 50U (bottom track) of Nt.CviPII in HCT116 (E) Venn diagram showing overlap of peaks in various amounts of Nt.CviPII in the universal NicE-seq (UniNicE-seq) reaction in HCT116 cells.

    Journal: bioRxiv

    Article Title: Universal NicE-seq for high resolution accessible chromatin profiling for formaldehyde fixed and FFPE tissues

    doi: 10.1101/2020.04.27.064592

    Figure Lengend Snippet: Addition of 5mdCTP in UniNicE-seq work flow. (A) Schematic diagram showing Nt.CviPII is blocked by 5-methylcytosine in recognition (CCD) sequence. During nick translation 5mdCTP can be incorporated at one or both cytosine position. Nucleotide mixtures containing biotinylated dCTP and 5mdCTP would allow both labels at CCD sites. Lower panel shows blocking of nicking by the presence of 5mC at CCD sites. After the nicking reaction the denatured oligonucleotides were resolved in a urea-acrylamide denaturing gel by electrophoresis. C is control input oligos, − or + indicate absence or presence of Nt.CviPII in the reaction. mC and btC represents 5-methylcytosine and biotinylated-cytosine respectively. Nicked oligonucleotide migrates faster on the denaturing urea gel. (B) Schematic diagram of UniNicE-seq method for accessible chromatin library preparation. (C) Substitution of dATP by Texas Red conjugated dATP will allow accessible chromatin visualization in the nucleus. (D) IGV screen shot of the normalized read density of UniNicE-seq using 2.5U (top track), 25U (middle track) and 50U (bottom track) of Nt.CviPII in HCT116 (E) Venn diagram showing overlap of peaks in various amounts of Nt.CviPII in the universal NicE-seq (UniNicE-seq) reaction in HCT116 cells.

    Article Snippet: Open chromatin DNA was labeled with biotin by incubating the nuclei in presence of 2.5 U of Nt.CviPII (NEB, R0626S), 50 U of DNA polymerase I (NEB, M0209S) and 30 μM of each dGTP and dTTP, 24 μM of 5mdCTP (NEB, NO356S) and dATP 24 μM plus 6 μM of biotin-14-dATP (Invitrogen, 19524016) and 6 μM of biotin-14-dCTP (Invitrogen, 19518018) at 37°C for 2 hrs with occasional mixing.

    Techniques: Sequencing, Nick Translation, Blocking Assay, Nucleic Acid Electrophoresis

    Cytogenetic analyses of C. roseus on mitotic spreads prepared from root tip cells . (A–C) Mitotic spreads of C. roseus stained with silver nitrate. (A) Prophase revealing a single nucleolus. (B) Prometaphase revealing a single pair of homologous chromosomes associated to the nucleolus. (C) Prometaphase with an already disorganized nucleolus and with the NORs restricted to a single chromosome pair. (D) Fluorescent staining with AD and DAPI of a prometaphase. (E) C-banding and staining with PI and DAPI of a prometaphase, highlighting the NORs in red. (F) Fluorescence in situ hybridization of rDNA of a metaphase. The probe (pTa71) was labelled with biotin-14-dATP and detected with fluorescein-avidin D, the chromosomes were counterstained with DAPI and the NORs are highlighted in green. Arrow indicates chromosome 8. (G) Catharanthus roseus karyotype. Chromosomes are aligned by the centromeric region and are ordered from 1 to 8, from the larger to the smaller, according to the morphometric analysis. (H) Ideogram of C. roseus chromosomes. Centromeres, constrictions, bands and NORs are depicted in the picture; the small arm of chromosome 8 is represented by a dashed line since it is usually not visible. Scale bars = 5 μm.

    Journal: AoB Plants

    Article Title: Cytogenetic characterization and genome size of the medicinal plant Catharanthus roseus (L.) G. Don

    doi: 10.1093/aobpla/pls002

    Figure Lengend Snippet: Cytogenetic analyses of C. roseus on mitotic spreads prepared from root tip cells . (A–C) Mitotic spreads of C. roseus stained with silver nitrate. (A) Prophase revealing a single nucleolus. (B) Prometaphase revealing a single pair of homologous chromosomes associated to the nucleolus. (C) Prometaphase with an already disorganized nucleolus and with the NORs restricted to a single chromosome pair. (D) Fluorescent staining with AD and DAPI of a prometaphase. (E) C-banding and staining with PI and DAPI of a prometaphase, highlighting the NORs in red. (F) Fluorescence in situ hybridization of rDNA of a metaphase. The probe (pTa71) was labelled with biotin-14-dATP and detected with fluorescein-avidin D, the chromosomes were counterstained with DAPI and the NORs are highlighted in green. Arrow indicates chromosome 8. (G) Catharanthus roseus karyotype. Chromosomes are aligned by the centromeric region and are ordered from 1 to 8, from the larger to the smaller, according to the morphometric analysis. (H) Ideogram of C. roseus chromosomes. Centromeres, constrictions, bands and NORs are depicted in the picture; the small arm of chromosome 8 is represented by a dashed line since it is usually not visible. Scale bars = 5 μm.

    Article Snippet: Plasmid DNA was labelled by nick translation using biotin-14-dATP of the Bionick™ Labeling System (Invitrogen Life Technologies, Paisley, UK).

    Techniques: Staining, Fluorescence, In Situ Hybridization, Avidin-Biotin Assay

    SATB1-binding sites are localized to the base of chromatin loops. In situ hybridization with Jurkat nuclei ( top ), nuclear halo ( middle ), and nuclear matrix ( bottom ) was performed using in vivo SATB1-binding sequences, SBS-2, SBS-3, and SBS-11, and control DNA, pTP18 (for U2 snRNA) and pH5SB (for the 5S rRNA gene), as DNA probes. These probes were labeled with biotin-11-dATP. Total DNA was stained with propidium iodide ( red ), and specific sequence hybridization was detected with FITC-conjugated extravidin ( yellow-green ). Since most of the genomic DNA has been removed from the nuclear matrix preparations, no propidium iodide staining was detectable in the nuclear matrix. In situ hybridization signals to the 5S rRNA within the distended chromatin halo are indicated by a white arrow.

    Journal: The Journal of Cell Biology

    Article Title: The Genomic Sequences Bound to Special AT-rich Sequence-binding Protein 1 (SATB1) In Vivo in Jurkat T Cells Are Tightly Associated with the Nuclear Matrix at the Bases of the Chromatin Loops

    doi:

    Figure Lengend Snippet: SATB1-binding sites are localized to the base of chromatin loops. In situ hybridization with Jurkat nuclei ( top ), nuclear halo ( middle ), and nuclear matrix ( bottom ) was performed using in vivo SATB1-binding sequences, SBS-2, SBS-3, and SBS-11, and control DNA, pTP18 (for U2 snRNA) and pH5SB (for the 5S rRNA gene), as DNA probes. These probes were labeled with biotin-11-dATP. Total DNA was stained with propidium iodide ( red ), and specific sequence hybridization was detected with FITC-conjugated extravidin ( yellow-green ). Since most of the genomic DNA has been removed from the nuclear matrix preparations, no propidium iodide staining was detectable in the nuclear matrix. In situ hybridization signals to the 5S rRNA within the distended chromatin halo are indicated by a white arrow.

    Article Snippet: For fluorescence in situ DNA hybridization (FISH), the DNA probes were labeled with biotin-14-dATP ( GIBCO BRL ) using a BioPrime DNA labeling system (Life Technologies, Gaithersburg, MD).

    Techniques: Binding Assay, In Situ Hybridization, In Vivo, Labeling, Staining, Sequencing, Hybridization