on column dnase digestion  (Qiagen)


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    Structured Review

    Qiagen on column dnase digestion
    Summary of in vitro primer extension and <t>DNase</t> I and KMnO 4 footprinting. ( A ) Sequence of P vpsL from −60 to +30. Bold and underlined A with black arrow at +1 and bold G (+3) represent the TSS determined by primer extensions; the −10 element and the −35 region are labeled and boxed in green; sequences in bold and red denote the VpsR binding site. Protection sites from DNase I footprinting and hypersensitive sites are depicted as rectangular boxes and triangles, respectively, either above (non-template) or below (template) the sequences: gray, RNAP with or without c-di-GMP or VpsR; black, RNAP with VpsR and c-di-GMP; red, VpsR with or without c-di-GMP. The open transcription bubble detected using KMnO 4 footprinting is shown as separated ssDNA from position −11 to +2 with sites of KMnO 4 cleavage indicated as purple asterisks. ( B ) Summary of positions of protection and hypersensitive sites on non-template and template strand DNA.
    On Column Dnase Digestion, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 288 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "VpsR and cyclic di-GMP together drive transcription initiation to activate biofilm formation in Vibrio cholerae"

    Article Title: VpsR and cyclic di-GMP together drive transcription initiation to activate biofilm formation in Vibrio cholerae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky606

    Summary of in vitro primer extension and DNase I and KMnO 4 footprinting. ( A ) Sequence of P vpsL from −60 to +30. Bold and underlined A with black arrow at +1 and bold G (+3) represent the TSS determined by primer extensions; the −10 element and the −35 region are labeled and boxed in green; sequences in bold and red denote the VpsR binding site. Protection sites from DNase I footprinting and hypersensitive sites are depicted as rectangular boxes and triangles, respectively, either above (non-template) or below (template) the sequences: gray, RNAP with or without c-di-GMP or VpsR; black, RNAP with VpsR and c-di-GMP; red, VpsR with or without c-di-GMP. The open transcription bubble detected using KMnO 4 footprinting is shown as separated ssDNA from position −11 to +2 with sites of KMnO 4 cleavage indicated as purple asterisks. ( B ) Summary of positions of protection and hypersensitive sites on non-template and template strand DNA.
    Figure Legend Snippet: Summary of in vitro primer extension and DNase I and KMnO 4 footprinting. ( A ) Sequence of P vpsL from −60 to +30. Bold and underlined A with black arrow at +1 and bold G (+3) represent the TSS determined by primer extensions; the −10 element and the −35 region are labeled and boxed in green; sequences in bold and red denote the VpsR binding site. Protection sites from DNase I footprinting and hypersensitive sites are depicted as rectangular boxes and triangles, respectively, either above (non-template) or below (template) the sequences: gray, RNAP with or without c-di-GMP or VpsR; black, RNAP with VpsR and c-di-GMP; red, VpsR with or without c-di-GMP. The open transcription bubble detected using KMnO 4 footprinting is shown as separated ssDNA from position −11 to +2 with sites of KMnO 4 cleavage indicated as purple asterisks. ( B ) Summary of positions of protection and hypersensitive sites on non-template and template strand DNA.

    Techniques Used: In Vitro, Footprinting, Sequencing, Labeling, Binding Assay

    DNase I footprinting of P vpsL complexes on ( A ) nontemplate DNA and ( B ) template DNA. GA corresponds to G+A ladder. VpsR, c-di-GMP and/or RNAP are present as indicated. To the right of each gel image, a schematic indicates the −10 and −35 regions and the +1. The VpsR binding site is indicated as a dashed black line. DNase I protection regions and hypersensitive sites seen with the activated complex of RNAP, VpsR, c-di-GMP and DNA are depicted as black rectangles and horizontal arrows, respectively. The dashed red boxes indicate the regions of DNA where the protection/enhancement within and immediately adjacent to the VpsR binding site changes when comparing complexes containing RNAP with or without VpsR or c-di-GMP to the activated complex. (See text for details.)
    Figure Legend Snippet: DNase I footprinting of P vpsL complexes on ( A ) nontemplate DNA and ( B ) template DNA. GA corresponds to G+A ladder. VpsR, c-di-GMP and/or RNAP are present as indicated. To the right of each gel image, a schematic indicates the −10 and −35 regions and the +1. The VpsR binding site is indicated as a dashed black line. DNase I protection regions and hypersensitive sites seen with the activated complex of RNAP, VpsR, c-di-GMP and DNA are depicted as black rectangles and horizontal arrows, respectively. The dashed red boxes indicate the regions of DNA where the protection/enhancement within and immediately adjacent to the VpsR binding site changes when comparing complexes containing RNAP with or without VpsR or c-di-GMP to the activated complex. (See text for details.)

    Techniques Used: Footprinting, Binding Assay

    2) Product Images from "VpsR and cyclic di-GMP together drive transcription initiation to activate biofilm formation in Vibrio cholerae"

    Article Title: VpsR and cyclic di-GMP together drive transcription initiation to activate biofilm formation in Vibrio cholerae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky606

    Summary of in vitro primer extension and DNase I and KMnO 4 footprinting. ( A ) Sequence of P vpsL from −60 to +30. Bold and underlined A with black arrow at +1 and bold G (+3) represent the TSS determined by primer extensions; the −10 element and the −35 region are labeled and boxed in green; sequences in bold and red denote the VpsR binding site. Protection sites from DNase I footprinting and hypersensitive sites are depicted as rectangular boxes and triangles, respectively, either above (non-template) or below (template) the sequences: gray, RNAP with or without c-di-GMP or VpsR; black, RNAP with VpsR and c-di-GMP; red, VpsR with or without c-di-GMP. The open transcription bubble detected using KMnO 4 footprinting is shown as separated ssDNA from position −11 to +2 with sites of KMnO 4 cleavage indicated as purple asterisks. ( B ) Summary of positions of protection and hypersensitive sites on non-template and template strand DNA.
    Figure Legend Snippet: Summary of in vitro primer extension and DNase I and KMnO 4 footprinting. ( A ) Sequence of P vpsL from −60 to +30. Bold and underlined A with black arrow at +1 and bold G (+3) represent the TSS determined by primer extensions; the −10 element and the −35 region are labeled and boxed in green; sequences in bold and red denote the VpsR binding site. Protection sites from DNase I footprinting and hypersensitive sites are depicted as rectangular boxes and triangles, respectively, either above (non-template) or below (template) the sequences: gray, RNAP with or without c-di-GMP or VpsR; black, RNAP with VpsR and c-di-GMP; red, VpsR with or without c-di-GMP. The open transcription bubble detected using KMnO 4 footprinting is shown as separated ssDNA from position −11 to +2 with sites of KMnO 4 cleavage indicated as purple asterisks. ( B ) Summary of positions of protection and hypersensitive sites on non-template and template strand DNA.

    Techniques Used: In Vitro, Footprinting, Sequencing, Labeling, Binding Assay

    DNase I footprinting of P vpsL complexes on ( A ) nontemplate DNA and ( B ) template DNA. GA corresponds to G+A ladder. VpsR, c-di-GMP and/or RNAP are present as indicated. To the right of each gel image, a schematic indicates the −10 and −35 regions and the +1. The VpsR binding site is indicated as a dashed black line. DNase I protection regions and hypersensitive sites seen with the activated complex of RNAP, VpsR, c-di-GMP and DNA are depicted as black rectangles and horizontal arrows, respectively. The dashed red boxes indicate the regions of DNA where the protection/enhancement within and immediately adjacent to the VpsR binding site changes when comparing complexes containing RNAP with or without VpsR or c-di-GMP to the activated complex. (See text for details.)
    Figure Legend Snippet: DNase I footprinting of P vpsL complexes on ( A ) nontemplate DNA and ( B ) template DNA. GA corresponds to G+A ladder. VpsR, c-di-GMP and/or RNAP are present as indicated. To the right of each gel image, a schematic indicates the −10 and −35 regions and the +1. The VpsR binding site is indicated as a dashed black line. DNase I protection regions and hypersensitive sites seen with the activated complex of RNAP, VpsR, c-di-GMP and DNA are depicted as black rectangles and horizontal arrows, respectively. The dashed red boxes indicate the regions of DNA where the protection/enhancement within and immediately adjacent to the VpsR binding site changes when comparing complexes containing RNAP with or without VpsR or c-di-GMP to the activated complex. (See text for details.)

    Techniques Used: Footprinting, Binding Assay

    3) Product Images from "Proteomic Analysis of the Quorum-Sensing Regulon in Pantoea stewartii and Identification of Direct Targets of EsaR"

    Article Title: Proteomic Analysis of the Quorum-Sensing Regulon in Pantoea stewartii and Identification of Direct Targets of EsaR

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01744-13

    DNase I footprinting assay of EsaR binding sites in the noncoding strand of P dkgA , coding strand of P glpF , and noncoding strand of P lrhA . (A to C) Capillary electrophoresis of FAM-labeled DNA fragments P dkgA (A), P glpF (B), and P lrhA (C) from DNase I
    Figure Legend Snippet: DNase I footprinting assay of EsaR binding sites in the noncoding strand of P dkgA , coding strand of P glpF , and noncoding strand of P lrhA . (A to C) Capillary electrophoresis of FAM-labeled DNA fragments P dkgA (A), P glpF (B), and P lrhA (C) from DNase I

    Techniques Used: Footprinting, Binding Assay, Electrophoresis, Labeling

    Nested deletion EMSA analysis of EsaR direct targets. (A to C) The region protected by DNase I digestion in P dkgA (A), P glpF (B), and P lrhA (C) is the gray-shaded sequence (5′ to 3′), and the underlined bases are the 20-bp EsaR binding
    Figure Legend Snippet: Nested deletion EMSA analysis of EsaR direct targets. (A to C) The region protected by DNase I digestion in P dkgA (A), P glpF (B), and P lrhA (C) is the gray-shaded sequence (5′ to 3′), and the underlined bases are the 20-bp EsaR binding

    Techniques Used: Sequencing, Binding Assay

    4) Product Images from "Expression of the Gonococcal Global Regulatory Protein Fur and Genes Encompassing the Fur and Iron Regulon during In Vitro and In Vivo Infection in Women "

    Article Title: Expression of the Gonococcal Global Regulatory Protein Fur and Genes Encompassing the Fur and Iron Regulon during In Vitro and In Vivo Infection in Women

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01830-07

    DNase I footprinting analysis of N. gonorrhoeae Fur protein with gonococcal fur and tonB promoter/operator regions. (A) DNase I footprint analysis of the fur promoter operator region. Radiolabeled DNA was incubated with increasing concentrations of Fur
    Figure Legend Snippet: DNase I footprinting analysis of N. gonorrhoeae Fur protein with gonococcal fur and tonB promoter/operator regions. (A) DNase I footprint analysis of the fur promoter operator region. Radiolabeled DNA was incubated with increasing concentrations of Fur

    Techniques Used: Footprinting, Incubation

    Related Articles

    Transduction:

    Article Title: DDX5 Regulates DNA Replication And Is Required For Cell Proliferation In A Subset Of Breast Cancer Cells
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    Clone Assay:

    Article Title: Mutations in Kelch-like 3 and Cullin 3 cause hypertension and electrolyte abnormalities
    Article Snippet: Products were cloned into the pcDNA6.2/GW/D-TOPO mammalian expression vector (Invitrogen), and plasmids were purified (QIAprep, Qiagen) and sequenced. .. RNA was isolated using RNeasy with DNase on-column digestion (Qiagen).

    Centrifugation:

    Article Title: VpsR and cyclic di-GMP together drive transcription initiation to activate biofilm formation in Vibrio cholerae
    Article Snippet: .. Cells were harvested by centrifugation and RNA was extracted using the RNeasy kit (Qiagen), and an on-column DNase I digestion (Qiagen) was performed according to manufacturer's instructions. ..

    Amplification:

    Article Title: Mutations in Kelch-like 3 and Cullin 3 cause hypertension and electrolyte abnormalities
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    DNA Synthesis:

    Article Title: Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge
    Article Snippet: Aqueous chloroform fractions were mixed with equal volumes of 70% ethanol/30% water and RNA further purified with RNeasy Mini spin columns (Qiagen) and on-column DNase-digestion (Qiagen). .. 1.5 μg of total RNA was used for complementary DNA synthesis using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). cDNAs were analyzed by qPCR using a Power SYBR Green PCR Master mix on the Quant Studio 6 Flex Real-Time PCR system (Applied Biosystems).

    Quantitative RT-PCR:

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    Article Snippet: .. RNA isolation, cDNA preparation, and RT-qPCR Total RNA was extracted and purified from whole-cell lysates using the RNeasy Mini Kit with on-column DNase digestion (Qiagen, Hilden, Germany; Cat. No. 74106). .. RNA concentration was determined on a NanoDrop 1000 (Thermo Fisher Scientific), and 1 µg of RNA used as a template for cDNA library preparation using the Maxima First Strand cDNA synthesis Kit for RT-qPCR (Thermo Fisher Scientific, Cat. No. K1641).

    SYBR Green Assay:

    Article Title: DDX5 Regulates DNA Replication And Is Required For Cell Proliferation In A Subset Of Breast Cancer Cells
    Article Snippet: RNA was prepared from siRNA transfected HCT116 cultures at the indicated time points post-siRNA transfection using the RNeasy Mini Kit (Qiagen cat. # 74104) including on-column DNase digestion (Qiagen cat. # 79254) and eluted in the supplied RNase-free water. .. Each Q-PCR reaction was prepared using 2μL of 1-to-20 diluted cDNA and 13μL LightCycler 480 SYBR Green I Master Mix (Roche #04887352001) and were performed in 384-well plates using the LightCycler 480 (Roche) as per manufacturer’s instructions.

    Article Title: Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge
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    Article Title: Targeting AURKA-CDC25C axis to induce synthetic lethality in ARID1A-deficient colorectal cancer cells
    Article Snippet: Total cellular RNA was harvested with the RNeasy Mini Kit, followed by on-column DNAse digestion (Qiagen, Hilden, Germany). .. AURKA transcription level was determined with SYBR Green Supermix (Bio-Rad, Hercules, CA) with the primer sequences as: forward primer 5’-CAGTACATGCTCCATCTTCCAG-3’ and reverse primer 5’-AAAGAACTCCAAGGCTCCAG-3’ (Integrated DNA Technologies).

    Article Title: ICE1 promotes the link between splicing and nonsense-mediated mRNA decay
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    Article Title: ICE1 promotes the link between splicing and nonsense-mediated mRNA decay
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    Article Title: Targeting AURKA-CDC25C axis to induce synthetic lethality in ARID1A-deficient colorectal cancer cells
    Article Snippet: RT and qPCR Total cellular RNA was harvested with the RNeasy Mini Kit, followed by on-column DNAse digestion (Qiagen, Hilden, Germany). .. AURKA transcription level was determined with SYBR Green Supermix (Bio-Rad, Hercules, CA) with the primer sequences as: forward primer 5’-CAGTACATGCTCCATCTTCCAG-3’ and reverse primer 5’-AAAGAACTCCAAGGCTCCAG-3’ (Integrated DNA Technologies).

    Random Hexamer Labeling:

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    Expressing:

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    Article Title: Mutations in Kelch-like 3 and Cullin 3 cause hypertension and electrolyte abnormalities
    Article Snippet: Products were cloned into the pcDNA6.2/GW/D-TOPO mammalian expression vector (Invitrogen), and plasmids were purified (QIAprep, Qiagen) and sequenced. .. RNA was isolated using RNeasy with DNase on-column digestion (Qiagen).

    Article Title: Inactivating hepatic follistatin alleviates hyperglycemia
    Article Snippet: RNA was extracted using TRI reagent (Sigma) and then subjected to a cleanup step using an RNeasy Mini Kit with on-column DNase digestion (Qiagen). .. Gene expression profiles were obtained using Affymetrix MoGene-2_0-st Chip in Genetics Core facility of Boston Children’s Hospital.

    Splicing Assay:

    Article Title: Mutations in Kelch-like 3 and Cullin 3 cause hypertension and electrolyte abnormalities
    Article Snippet: Paragraph title: Splicing assay ... RNA was isolated using RNeasy with DNase on-column digestion (Qiagen).

    Transfection:

    Article Title: DDX5 Regulates DNA Replication And Is Required For Cell Proliferation In A Subset Of Breast Cancer Cells
    Article Snippet: .. RNA was prepared from siRNA transfected HCT116 cultures at the indicated time points post-siRNA transfection using the RNeasy Mini Kit (Qiagen cat. # 74104) including on-column DNase digestion (Qiagen cat. # 79254) and eluted in the supplied RNase-free water. .. RNA from shRNA SK-BR-3 cultures was prepared from cells 8 days post-shRNA transduction.

    Article Title: ICE1 promotes the link between splicing and nonsense-mediated mRNA decay
    Article Snippet: Metabolic labeling and mRNA half-life quantification To determine specific mRNA half-life measurements during ICE1 and UPF1 depletion, HEK-293 cells were reverse transfected with a gene-specific or non-targeting control siRNA for 72 hr. .. Cells were then immediately harvested and RNA was isolated and column purified using the RNeasy Mini Kit with on-column DNase digestion (Qiagen, Hilden, Germany; Cat. No. 74106).

    Article Title: Mutations in Kelch-like 3 and Cullin 3 cause hypertension and electrolyte abnormalities
    Article Snippet: HEK293 cells were transfected independently with each plasmid using Lipofectamine 2000 (Invitrogen) and harvested ~24 hours post-transfection. .. RNA was isolated using RNeasy with DNase on-column digestion (Qiagen).

    Sequencing:

    Article Title: Mutations in Kelch-like 3 and Cullin 3 cause hypertension and electrolyte abnormalities
    Article Snippet: Splicing assay A 3782 bp segment of CUL3 , extending from 287 bp proximal to exon 8 to 327 bp distal to exon 10, was amplified by PCR (Advantage 2 polymerase, Clontech) from genomic DNA of nine PHAII patients with different CUL3 mutations and one subject with wild-type CUL3 sequence. .. RNA was isolated using RNeasy with DNase on-column digestion (Qiagen).

    Cell Culture:

    Article Title: Targeting RNA structure in SMN2 reverses spinal muscular atrophy molecular phenotypes
    Article Snippet: Total RNA was extracted using the RNeasy mini kit with on-column DNase digestion (Qiagen). .. Between 0.5 and 1 μg of RNA were used for reverse transcription with Super Script II (Invitrogen) or the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), using random hexamers (cultured cells) or a gene-specific primer 5′-CAGCGTAAGTGATGTCCACCT-3′ ( Drosophila ).

    Article Title: ICE1 promotes the link between splicing and nonsense-mediated mRNA decay
    Article Snippet: Cells were then immediately harvested and RNA was isolated and column purified using the RNeasy Mini Kit with on-column DNase digestion (Qiagen, Hilden, Germany; Cat. No. 74106). .. In addition, Drosophila S2 cells (ThermoFisher Scientific, Cat. No. R690-07) were cultured at ambient temperature for 24 hr in Sf-900 media (ThermoFisher Scientific, Cat. No. 10967032) containing 0.1 mM 5-EU, and RNA was isolated and processed as with the human cells for use as a spike-in control.

    Generated:

    Article Title: VpsR and cyclic di-GMP together drive transcription initiation to activate biofilm formation in Vibrio cholerae
    Article Snippet: Primer extension analyses of RNA generated in vitro were performed according to manufacturer’s instructions (Promega) by using AMV Reverse Transcriptase. .. Cells were harvested by centrifugation and RNA was extracted using the RNeasy kit (Qiagen), and an on-column DNase I digestion (Qiagen) was performed according to manufacturer's instructions.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Targeting RNA structure in SMN2 reverses spinal muscular atrophy molecular phenotypes
    Article Snippet: Paragraph title: Reverse transcription (RT)-PCR ... Total RNA was extracted using the RNeasy mini kit with on-column DNase digestion (Qiagen).

    In Vivo:

    Article Title: VpsR and cyclic di-GMP together drive transcription initiation to activate biofilm formation in Vibrio cholerae
    Article Snippet: In vivo RNA was obtained from WN310 containing pMLH17 and pCMW75 or pCMW98 grown in LB with 100 μg/ml ampicillin and 100 μg/ml kanamycin at 37°C with shaking at 220 rpm to an OD600 of ∼0.5. .. Cells were harvested by centrifugation and RNA was extracted using the RNeasy kit (Qiagen), and an on-column DNase I digestion (Qiagen) was performed according to manufacturer's instructions.

    RNA Sequencing Assay:

    Article Title: GKAP Acts as a Genetic Modulator of NMDAR Signaling to Govern Invasive Tumor Growth
    Article Snippet: Paragraph title: RNA-Seq Sample Collection (B6/C3H/MK801) ... On-column DNase digestion (QIAGEN) were performed for all samples during RNA extraction according to manufacturer’s protocol.

    Isolation:

    Article Title: Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge
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    Article Title: ICE1 promotes the link between splicing and nonsense-mediated mRNA decay
    Article Snippet: .. Cells were then immediately harvested and RNA was isolated and column purified using the RNeasy Mini Kit with on-column DNase digestion (Qiagen, Hilden, Germany; Cat. No. 74106). .. In addition, Drosophila S2 cells (ThermoFisher Scientific, Cat. No. R690-07) were cultured at ambient temperature for 24 hr in Sf-900 media (ThermoFisher Scientific, Cat. No. 10967032) containing 0.1 mM 5-EU, and RNA was isolated and processed as with the human cells for use as a spike-in control.

    Article Title: ICE1 promotes the link between splicing and nonsense-mediated mRNA decay
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    Article Title: Mutations in Kelch-like 3 and Cullin 3 cause hypertension and electrolyte abnormalities
    Article Snippet: .. RNA was isolated using RNeasy with DNase on-column digestion (Qiagen). .. The spliced expression products were assessed by reverse transcription with oligo(dT) priming (Superscript III RT, Invitrogen) followed by PCR with vector-specific and CUL3 -specific primers.

    Article Title: ICE1 promotes the link between splicing and nonsense-mediated mRNA decay
    Article Snippet: .. RNA isolation, cDNA preparation, and RT-qPCR Total RNA was extracted and purified from whole-cell lysates using the RNeasy Mini Kit with on-column DNase digestion (Qiagen, Hilden, Germany; Cat. No. 74106). .. RNA concentration was determined on a NanoDrop 1000 (Thermo Fisher Scientific), and 1 µg of RNA used as a template for cDNA library preparation using the Maxima First Strand cDNA synthesis Kit for RT-qPCR (Thermo Fisher Scientific, Cat. No. K1641).

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    Labeling:

    Article Title: ICE1 promotes the link between splicing and nonsense-mediated mRNA decay
    Article Snippet: Paragraph title: Metabolic labeling and mRNA half-life quantification ... Cells were then immediately harvested and RNA was isolated and column purified using the RNeasy Mini Kit with on-column DNase digestion (Qiagen, Hilden, Germany; Cat. No. 74106).

    Purification:

    Article Title: Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge
    Article Snippet: .. Aqueous chloroform fractions were mixed with equal volumes of 70% ethanol/30% water and RNA further purified with RNeasy Mini spin columns (Qiagen) and on-column DNase-digestion (Qiagen). .. TRIzol phases were saved for further isolation of genomic and mitochondrial DNA.

    Article Title: ICE1 promotes the link between splicing and nonsense-mediated mRNA decay
    Article Snippet: .. Cells were then immediately harvested and RNA was isolated and column purified using the RNeasy Mini Kit with on-column DNase digestion (Qiagen, Hilden, Germany; Cat. No. 74106). .. In addition, Drosophila S2 cells (ThermoFisher Scientific, Cat. No. R690-07) were cultured at ambient temperature for 24 hr in Sf-900 media (ThermoFisher Scientific, Cat. No. 10967032) containing 0.1 mM 5-EU, and RNA was isolated and processed as with the human cells for use as a spike-in control.

    Article Title: ICE1 promotes the link between splicing and nonsense-mediated mRNA decay
    Article Snippet: .. Total RNA was extracted and purified from whole-cell lysates using the RNeasy Mini Kit with on-column DNase digestion (Qiagen, Hilden, Germany; Cat. No. 74106). .. RNA concentration was determined on a NanoDrop 1000 (Thermo Fisher Scientific), and 1 µg of RNA used as a template for cDNA library preparation using the Maxima First Strand cDNA synthesis Kit for RT-qPCR (Thermo Fisher Scientific, Cat. No. K1641).

    Article Title: Mutations in Kelch-like 3 and Cullin 3 cause hypertension and electrolyte abnormalities
    Article Snippet: Products were cloned into the pcDNA6.2/GW/D-TOPO mammalian expression vector (Invitrogen), and plasmids were purified (QIAprep, Qiagen) and sequenced. .. RNA was isolated using RNeasy with DNase on-column digestion (Qiagen).

    Article Title: ICE1 promotes the link between splicing and nonsense-mediated mRNA decay
    Article Snippet: .. RNA isolation, cDNA preparation, and RT-qPCR Total RNA was extracted and purified from whole-cell lysates using the RNeasy Mini Kit with on-column DNase digestion (Qiagen, Hilden, Germany; Cat. No. 74106). .. RNA concentration was determined on a NanoDrop 1000 (Thermo Fisher Scientific), and 1 µg of RNA used as a template for cDNA library preparation using the Maxima First Strand cDNA synthesis Kit for RT-qPCR (Thermo Fisher Scientific, Cat. No. K1641).

    Polymerase Chain Reaction:

    Article Title: Targeting RNA structure in SMN2 reverses spinal muscular atrophy molecular phenotypes
    Article Snippet: Total RNA was extracted using the RNeasy mini kit with on-column DNase digestion (Qiagen). .. A total of 100 ng of cDNA were used as template in semi-quantitative PCR with GoTaq polymerase (cultured cells; Promega) or 2×PCR Super Master Mix ( Drosophila , Biotool).

    Article Title: DDX5 Regulates DNA Replication And Is Required For Cell Proliferation In A Subset Of Breast Cancer Cells
    Article Snippet: RNA was prepared from siRNA transfected HCT116 cultures at the indicated time points post-siRNA transfection using the RNeasy Mini Kit (Qiagen cat. # 74104) including on-column DNase digestion (Qiagen cat. # 79254) and eluted in the supplied RNase-free water. .. The cDNA used for Q-PCR was prepared from 1μg each RNA sample using TaqMan Reverse Transcription Reagents (Applied Biosystems #N808-0234) with random hexamer priming in a GeneAmp PCR system 9700 thermocycler.

    Article Title: Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge
    Article Snippet: Aqueous chloroform fractions were mixed with equal volumes of 70% ethanol/30% water and RNA further purified with RNeasy Mini spin columns (Qiagen) and on-column DNase-digestion (Qiagen). .. 1.5 μg of total RNA was used for complementary DNA synthesis using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). cDNAs were analyzed by qPCR using a Power SYBR Green PCR Master mix on the Quant Studio 6 Flex Real-Time PCR system (Applied Biosystems).

    Article Title: Mutations in Kelch-like 3 and Cullin 3 cause hypertension and electrolyte abnormalities
    Article Snippet: Splicing assay A 3782 bp segment of CUL3 , extending from 287 bp proximal to exon 8 to 327 bp distal to exon 10, was amplified by PCR (Advantage 2 polymerase, Clontech) from genomic DNA of nine PHAII patients with different CUL3 mutations and one subject with wild-type CUL3 sequence. .. RNA was isolated using RNeasy with DNase on-column digestion (Qiagen).

    cDNA Library Assay:

    Article Title: ICE1 promotes the link between splicing and nonsense-mediated mRNA decay
    Article Snippet: Total RNA was extracted and purified from whole-cell lysates using the RNeasy Mini Kit with on-column DNase digestion (Qiagen, Hilden, Germany; Cat. No. 74106). .. RNA concentration was determined on a NanoDrop 1000 (Thermo Fisher Scientific), and 1 µg of RNA used as a template for cDNA library preparation using the Maxima First Strand cDNA synthesis Kit for RT-qPCR (Thermo Fisher Scientific, Cat. No. K1641).

    Article Title: ICE1 promotes the link between splicing and nonsense-mediated mRNA decay
    Article Snippet: RNA isolation, cDNA preparation, and RT-qPCR Total RNA was extracted and purified from whole-cell lysates using the RNeasy Mini Kit with on-column DNase digestion (Qiagen, Hilden, Germany; Cat. No. 74106). .. RNA concentration was determined on a NanoDrop 1000 (Thermo Fisher Scientific), and 1 µg of RNA used as a template for cDNA library preparation using the Maxima First Strand cDNA synthesis Kit for RT-qPCR (Thermo Fisher Scientific, Cat. No. K1641).

    Mouse Assay:

    Article Title: GKAP Acts as a Genetic Modulator of NMDAR Signaling to Govern Invasive Tumor Growth
    Article Snippet: Control mice were either not treated, or treated with normal saline (“sal”) for 3 days prior to sacrifice; MK801-treated mice were treated for 3 days prior to sacrifice. .. On-column DNase digestion (QIAGEN) were performed for all samples during RNA extraction according to manufacturer’s protocol.

    Article Title: Unravelling Endogenous MicroRNA System Dysfunction as a New Pathophysiological Mechanism in Machado-Joseph Disease
    Article Snippet: Striatum of mice were dissected and stored at −80°C until RNA isolation. .. All samples were submitted to on-column DNase I digestion (QIAGEN) during isolation.

    Chromatin Immunoprecipitation:

    Article Title: Inactivating hepatic follistatin alleviates hyperglycemia
    Article Snippet: RNA was extracted using TRI reagent (Sigma) and then subjected to a cleanup step using an RNeasy Mini Kit with on-column DNase digestion (Qiagen). .. Gene expression profiles were obtained using Affymetrix MoGene-2_0-st Chip in Genetics Core facility of Boston Children’s Hospital.

    Plasmid Preparation:

    Article Title: Mutations in Kelch-like 3 and Cullin 3 cause hypertension and electrolyte abnormalities
    Article Snippet: HEK293 cells were transfected independently with each plasmid using Lipofectamine 2000 (Invitrogen) and harvested ~24 hours post-transfection. .. RNA was isolated using RNeasy with DNase on-column digestion (Qiagen).

    Real-time Polymerase Chain Reaction:

    Article Title: DDX5 Regulates DNA Replication And Is Required For Cell Proliferation In A Subset Of Breast Cancer Cells
    Article Snippet: Paragraph title: Quantitative PCR analysis ... RNA was prepared from siRNA transfected HCT116 cultures at the indicated time points post-siRNA transfection using the RNeasy Mini Kit (Qiagen cat. # 74104) including on-column DNase digestion (Qiagen cat. # 79254) and eluted in the supplied RNase-free water.

    Article Title: Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge
    Article Snippet: Aqueous chloroform fractions were mixed with equal volumes of 70% ethanol/30% water and RNA further purified with RNeasy Mini spin columns (Qiagen) and on-column DNase-digestion (Qiagen). .. 1.5 μg of total RNA was used for complementary DNA synthesis using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). cDNAs were analyzed by qPCR using a Power SYBR Green PCR Master mix on the Quant Studio 6 Flex Real-Time PCR system (Applied Biosystems).

    Article Title: Targeting AURKA-CDC25C axis to induce synthetic lethality in ARID1A-deficient colorectal cancer cells
    Article Snippet: Paragraph title: RT and qPCR ... Total cellular RNA was harvested with the RNeasy Mini Kit, followed by on-column DNAse digestion (Qiagen, Hilden, Germany).

    Article Title: Targeting AURKA-CDC25C axis to induce synthetic lethality in ARID1A-deficient colorectal cancer cells
    Article Snippet: .. RT and qPCR Total cellular RNA was harvested with the RNeasy Mini Kit, followed by on-column DNAse digestion (Qiagen, Hilden, Germany). .. RT was performed with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific).

    RNA Extraction:

    Article Title: GKAP Acts as a Genetic Modulator of NMDAR Signaling to Govern Invasive Tumor Growth
    Article Snippet: .. On-column DNase digestion (QIAGEN) were performed for all samples during RNA extraction according to manufacturer’s protocol. .. Antibodies GluN2b (Pierce #PA3-105) for IP, WB and IHC, (NeuroMab #75-097, 75-101) for WB; GKAP (Sigma # PRS4623) for IHC, IP, and WB 1:1000 in mouse, (R & D #MAB7296) for IHC in human PDAC; FMRP (Cell Signaling, #4317); HSF1 (Santa Cruz, #sc-9144) for ChIP, (Cell Signaling, #4356) for IHC 1:50.

    shRNA:

    Article Title: DDX5 Regulates DNA Replication And Is Required For Cell Proliferation In A Subset Of Breast Cancer Cells
    Article Snippet: RNA was prepared from siRNA transfected HCT116 cultures at the indicated time points post-siRNA transfection using the RNeasy Mini Kit (Qiagen cat. # 74104) including on-column DNase digestion (Qiagen cat. # 79254) and eluted in the supplied RNase-free water. .. RNA from shRNA SK-BR-3 cultures was prepared from cells 8 days post-shRNA transduction.

    In Vitro:

    Article Title: VpsR and cyclic di-GMP together drive transcription initiation to activate biofilm formation in Vibrio cholerae
    Article Snippet: A sample (5 μl) of the in vitro transcription reaction was added to 6 μl of primer mixture containing 2 × AMV Primer Extension buffer and 2 pmol 32 P-labeled primer, which annealed +103 bp downstream of the +1 transcriptional start site. .. Cells were harvested by centrifugation and RNA was extracted using the RNeasy kit (Qiagen), and an on-column DNase I digestion (Qiagen) was performed according to manufacturer's instructions.

    Homogenization:

    Article Title: GKAP Acts as a Genetic Modulator of NMDAR Signaling to Govern Invasive Tumor Growth
    Article Snippet: Tissue homogenization was done on TIssueLyser II (QIAGEN) in pre-cooled cassettes, with disposable beads in 700 μl Qiazol (from the miRNeasy kit)/ tumor. .. On-column DNase digestion (QIAGEN) were performed for all samples during RNA extraction according to manufacturer’s protocol.

    Spectrophotometry:

    Article Title: Unravelling Endogenous MicroRNA System Dysfunction as a New Pathophysiological Mechanism in Machado-Joseph Disease
    Article Snippet: All samples were submitted to on-column DNase I digestion (QIAGEN) during isolation. .. Total amount of RNA was quantified by optical density (OD) using a NanoDrop 2000 Spectrophotometer (Thermo Scientific) and RNA was stored at −80°C.

    Concentration Assay:

    Article Title: ICE1 promotes the link between splicing and nonsense-mediated mRNA decay
    Article Snippet: Total RNA was extracted and purified from whole-cell lysates using the RNeasy Mini Kit with on-column DNase digestion (Qiagen, Hilden, Germany; Cat. No. 74106). .. RNA concentration was determined on a NanoDrop 1000 (Thermo Fisher Scientific), and 1 µg of RNA used as a template for cDNA library preparation using the Maxima First Strand cDNA synthesis Kit for RT-qPCR (Thermo Fisher Scientific, Cat. No. K1641).

    Article Title: ICE1 promotes the link between splicing and nonsense-mediated mRNA decay
    Article Snippet: RNA isolation, cDNA preparation, and RT-qPCR Total RNA was extracted and purified from whole-cell lysates using the RNeasy Mini Kit with on-column DNase digestion (Qiagen, Hilden, Germany; Cat. No. 74106). .. RNA concentration was determined on a NanoDrop 1000 (Thermo Fisher Scientific), and 1 µg of RNA used as a template for cDNA library preparation using the Maxima First Strand cDNA synthesis Kit for RT-qPCR (Thermo Fisher Scientific, Cat. No. K1641).

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    Qiagen rnase free dnase set
    Rnase Free Dnase Set, supplied by Qiagen, used in various techniques. Bioz Stars score: 90/100, based on 962 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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