dnaase  (Thermo Fisher)


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    Structured Review

    Thermo Fisher dnaase
    CRISPRd Disrupts Nanog <t>DNA</t> Binding and Identifies an Autoinhibitory Element in the Nanog Promoter (A) Top: a diagram ofthe Nanog locus with the genomic region shown below indicated by the orange box. Bottom: ChIP-seq data for Nanog show a binding site located 4.9 kb upstream of its own transcription start site. Position of the targeting sgRNA shown in red. The red box denotes open chromatin identified by ENCODE <t>DNase-seq</t> (Experiment number: ENCSR000CMW). (B) dCas9 ChIP-qPCR measurements for cells expressing dCas9, and the indicated sgRNA shows recruitment of dCas9 to the Nanog binding site. (C) Nanog ChIP-qPCR shows that Nanog binding to its own promoter is decreased by 2 out of 3 targeting sgRNAs. (D) qRT-PCR of Nanog in cells expressing the sgRNA targeting the Nanog binding site shows increased expression of Nanog mRNA. (E)Immunoblot of Nanog in cells expressing the indicated sgRNA. (F)Quantification of three independent Nanog immunoblots as in (E). (G)Schematic representation of the negative feedback loop formed by Nanog binding to its own promoter. Bar plots show mean and associated standard error. ns, *, and *** denote p > 0.05, p
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    1) Product Images from "Reversible Disruption of Specific Transcription Factor-DNA Interactions Using CRISPR/Cas9"

    Article Title: Reversible Disruption of Specific Transcription Factor-DNA Interactions Using CRISPR/Cas9

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2019.04.011

    CRISPRd Disrupts Nanog DNA Binding and Identifies an Autoinhibitory Element in the Nanog Promoter (A) Top: a diagram ofthe Nanog locus with the genomic region shown below indicated by the orange box. Bottom: ChIP-seq data for Nanog show a binding site located 4.9 kb upstream of its own transcription start site. Position of the targeting sgRNA shown in red. The red box denotes open chromatin identified by ENCODE DNase-seq (Experiment number: ENCSR000CMW). (B) dCas9 ChIP-qPCR measurements for cells expressing dCas9, and the indicated sgRNA shows recruitment of dCas9 to the Nanog binding site. (C) Nanog ChIP-qPCR shows that Nanog binding to its own promoter is decreased by 2 out of 3 targeting sgRNAs. (D) qRT-PCR of Nanog in cells expressing the sgRNA targeting the Nanog binding site shows increased expression of Nanog mRNA. (E)Immunoblot of Nanog in cells expressing the indicated sgRNA. (F)Quantification of three independent Nanog immunoblots as in (E). (G)Schematic representation of the negative feedback loop formed by Nanog binding to its own promoter. Bar plots show mean and associated standard error. ns, *, and *** denote p > 0.05, p
    Figure Legend Snippet: CRISPRd Disrupts Nanog DNA Binding and Identifies an Autoinhibitory Element in the Nanog Promoter (A) Top: a diagram ofthe Nanog locus with the genomic region shown below indicated by the orange box. Bottom: ChIP-seq data for Nanog show a binding site located 4.9 kb upstream of its own transcription start site. Position of the targeting sgRNA shown in red. The red box denotes open chromatin identified by ENCODE DNase-seq (Experiment number: ENCSR000CMW). (B) dCas9 ChIP-qPCR measurements for cells expressing dCas9, and the indicated sgRNA shows recruitment of dCas9 to the Nanog binding site. (C) Nanog ChIP-qPCR shows that Nanog binding to its own promoter is decreased by 2 out of 3 targeting sgRNAs. (D) qRT-PCR of Nanog in cells expressing the sgRNA targeting the Nanog binding site shows increased expression of Nanog mRNA. (E)Immunoblot of Nanog in cells expressing the indicated sgRNA. (F)Quantification of three independent Nanog immunoblots as in (E). (G)Schematic representation of the negative feedback loop formed by Nanog binding to its own promoter. Bar plots show mean and associated standard error. ns, *, and *** denote p > 0.05, p

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Western Blot

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    Thermo Fisher gfp
    Analysis of <t>pri-miRNA</t> folding and transcription. (A) Illustration of putative secondary structures for both artificial and endogenous mir–221 and mir-15b using Quikfold [ 38 , http://mfold.rna.albany.edu/?q=DINAMelt/Quickfold] with energy rules for RNA(3.0) and default settings. The location of primers used from amplifying the respective pri–miRNAs, the mature miR–sequences, and the cleavage sites (Drosha/Dgcr8 at the stem/duplex interface, Dicer at the duplex/loop interface) are indicated. (B) pri-mir-221 and <t>GFP</t> transcription levels two days post transfection analyzed by RT-qPCR, normalized against GAPDH and related to the negative control (mean ± standard deviation of three individual transfections).
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    Thermo Fisher dnase i
    TRIM17 invalidation induces BCL2A1 degradation and sensitizes melanoma cells to BRAF-targeted therapy.  a  Method outline depicting generation of TRIM17-depleted SK-MEL-28 cells by inducible CRISPR/Cas9 method and workflow of subsequent steps of analysis.  b  SK-MEL-28 cells expressing Cas9 and inducible sgRNAs against TRIM17 were treated for 72 h with 1 μg/ml doxycycline. Genomic DNA of cells was amplified by PCR, around the Cas9 cleavage sites targeted by the two sgTRIM17, and PCR products were analyzed by a T7 Endonuclease I assay. The presence of InDels was visualized by the digestion of the PCR products at the Cas9 cleavage sites by T7EI.  c  PLA was performed in SK-MEL-28 cells expressing DOX-inducible sgRNA against TRIM17, using anti-TRIM28 and anti-BCL2A1 antibodies. Note that the number of green bright spots indicating the very close proximity between endogenous TRIM28 and BCL2A1 proteins increases when expression of endogenous TRIM17 is inhibited by doxycycline treatment. Negative control was obtained by omitting the anti-BCL2A1 antibody. Scale bars, 10 μm.  d  The protein level of endogenous BCL2A1 was assessed by immunoblot using anti-BCL2A1 antibody in total protein extracts from non-transduced SK-MEL-28 cells left untreated or treated with 20 μM MG132 for 6 h, or from transduced cells expressing Cas9 alone or Cas9 together with inducible sgRNAs against BCL2A1 or mouse  bim  (negative control) and treated or not with 1 μg/ml doxycycline for 72 h, as indicated.  e  Two sgRNAs against  TRIM17  were induced in SK-MEL-28 cells by doxycycline treatment for 72 h and protein level of endogenous BCL2A1 was assessed by immunoblot using anti-BCL2A1 antibody.  f  SK-MEL-28 cells expressing Cas9 and the indicated sgRNAs were treated with 1 μg/ml doxycycline for 72 h and subsequently treated with 20 μM PLX4720 (or DMSO control) for 48 h. PLX4720-specific induced apoptosis (SIA) was assessed by annexin V staining and flow cytometry. Data are the means ± SEM of three independent experiments. **** p
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    Thermo Fisher rna
    Comparison of the growth rate of H3N2 A/Guangdong/ST798/2008 strains carrying either 526R or 526K PB2 in mixed cultures. ( a ) MDCK cells or ( b ) A549 cells were infected at an MOI of 0.001 with a mixture (1:1) of wild-type (WT) (PB2-526R) and variant (PB2 R526K) A/Guangdong/ST798/2008 and sequentially passaged four times. At each passage viral <t>RNA</t> was isolated from cell culture supernatants at 48 h post infection, and the PB2 gene amplified by RT-PCR and sequenced. The genetic code for WT PB2-526R is AGA; variant PB2 526K is AAA. The <t>DNA</t> sequence chromatograms shown are from one of the three MDCK ( a ) and one of the four A549 ( b ) experiments in which 526R outgrew 526K. Experiments were repeated four times.
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    Analysis of pri-miRNA folding and transcription. (A) Illustration of putative secondary structures for both artificial and endogenous mir–221 and mir-15b using Quikfold [ 38 , http://mfold.rna.albany.edu/?q=DINAMelt/Quickfold] with energy rules for RNA(3.0) and default settings. The location of primers used from amplifying the respective pri–miRNAs, the mature miR–sequences, and the cleavage sites (Drosha/Dgcr8 at the stem/duplex interface, Dicer at the duplex/loop interface) are indicated. (B) pri-mir-221 and GFP transcription levels two days post transfection analyzed by RT-qPCR, normalized against GAPDH and related to the negative control (mean ± standard deviation of three individual transfections).

    Journal: Biotechnology Journal

    Article Title: Endogenous microRNA clusters outperform chimeric sequence clusters in Chinese hamster ovary cells

    doi: 10.1002/biot.201300216

    Figure Lengend Snippet: Analysis of pri-miRNA folding and transcription. (A) Illustration of putative secondary structures for both artificial and endogenous mir–221 and mir-15b using Quikfold [ 38 , http://mfold.rna.albany.edu/?q=DINAMelt/Quickfold] with energy rules for RNA(3.0) and default settings. The location of primers used from amplifying the respective pri–miRNAs, the mature miR–sequences, and the cleavage sites (Drosha/Dgcr8 at the stem/duplex interface, Dicer at the duplex/loop interface) are indicated. (B) pri-mir-221 and GFP transcription levels two days post transfection analyzed by RT-qPCR, normalized against GAPDH and related to the negative control (mean ± standard deviation of three individual transfections).

    Article Snippet: 2.8 Quantitation of primary miRNA transcripts and GFP 800 ng of DNase I (Fermentas, Waltham, MA, USA) treated total RNA of each sample were denatured for 2 minutes at 72°C and then put on ice. cDNA was generated by the DyNAmo cDNA Synthesis Kit (Thermo Scientific, Pittsburgh PA), consisting of the M-MuLV RNase H+ reverse transcriptase and random hexamer primers.

    Techniques: Transfection, Quantitative RT-PCR, Negative Control, Standard Deviation

    Human TDP-43 displays strand dissociation activity in vitro Assay for strand displacement activity (adapted from Naeeni et al , 2012 ). RNA strand dissociation activity was measured as a change in relative FRET signal ( y -axis) over time ( x -axis) of two complementary, fluorescently labeled (Cy5 and Cy3, respectively) RNA oligos (annealed for 480 s at 37°C) following the addition of tenfold molar excess of a competitor oligo complementary to the Cy5-labeled RNA molecule. RNA molecules were excited at 535 nm, and FRET signal was recorded at 680 nm. Initial FRET fluorescence was set to zero and then measured every 15 s for 480 s. Data represent results from six to seven independent assays. Error bars, SEM calculated between replicates at each 15 s time point. The assay was done in the presence of Bovine Serum Albumin (BSA) protein (blue line); recombinant human Lupus Antigen (Human La) (red line), and recombinant human Tar-DNA Binding protein 43 (TDP-43) (green line).

    Journal: The EMBO Journal

    Article Title: TDP-1, the Caenorhabditis elegans ortholog of TDP-43, limits the accumulation of double-stranded RNA

    doi: 10.15252/embj.201488740

    Figure Lengend Snippet: Human TDP-43 displays strand dissociation activity in vitro Assay for strand displacement activity (adapted from Naeeni et al , 2012 ). RNA strand dissociation activity was measured as a change in relative FRET signal ( y -axis) over time ( x -axis) of two complementary, fluorescently labeled (Cy5 and Cy3, respectively) RNA oligos (annealed for 480 s at 37°C) following the addition of tenfold molar excess of a competitor oligo complementary to the Cy5-labeled RNA molecule. RNA molecules were excited at 535 nm, and FRET signal was recorded at 680 nm. Initial FRET fluorescence was set to zero and then measured every 15 s for 480 s. Data represent results from six to seven independent assays. Error bars, SEM calculated between replicates at each 15 s time point. The assay was done in the presence of Bovine Serum Albumin (BSA) protein (blue line); recombinant human Lupus Antigen (Human La) (red line), and recombinant human Tar-DNA Binding protein 43 (TDP-43) (green line).

    Article Snippet: DNA was removed from immunoprecipitated RNA using TURBO DNase (Invitrogen) and dissolved in nuclease-free water.

    Techniques: Activity Assay, In Vitro, Labeling, Fluorescence, Recombinant, Binding Assay

    TRIM17 invalidation induces BCL2A1 degradation and sensitizes melanoma cells to BRAF-targeted therapy.  a  Method outline depicting generation of TRIM17-depleted SK-MEL-28 cells by inducible CRISPR/Cas9 method and workflow of subsequent steps of analysis.  b  SK-MEL-28 cells expressing Cas9 and inducible sgRNAs against TRIM17 were treated for 72 h with 1 μg/ml doxycycline. Genomic DNA of cells was amplified by PCR, around the Cas9 cleavage sites targeted by the two sgTRIM17, and PCR products were analyzed by a T7 Endonuclease I assay. The presence of InDels was visualized by the digestion of the PCR products at the Cas9 cleavage sites by T7EI.  c  PLA was performed in SK-MEL-28 cells expressing DOX-inducible sgRNA against TRIM17, using anti-TRIM28 and anti-BCL2A1 antibodies. Note that the number of green bright spots indicating the very close proximity between endogenous TRIM28 and BCL2A1 proteins increases when expression of endogenous TRIM17 is inhibited by doxycycline treatment. Negative control was obtained by omitting the anti-BCL2A1 antibody. Scale bars, 10 μm.  d  The protein level of endogenous BCL2A1 was assessed by immunoblot using anti-BCL2A1 antibody in total protein extracts from non-transduced SK-MEL-28 cells left untreated or treated with 20 μM MG132 for 6 h, or from transduced cells expressing Cas9 alone or Cas9 together with inducible sgRNAs against BCL2A1 or mouse  bim  (negative control) and treated or not with 1 μg/ml doxycycline for 72 h, as indicated.  e  Two sgRNAs against  TRIM17  were induced in SK-MEL-28 cells by doxycycline treatment for 72 h and protein level of endogenous BCL2A1 was assessed by immunoblot using anti-BCL2A1 antibody.  f  SK-MEL-28 cells expressing Cas9 and the indicated sgRNAs were treated with 1 μg/ml doxycycline for 72 h and subsequently treated with 20 μM PLX4720 (or DMSO control) for 48 h. PLX4720-specific induced apoptosis (SIA) was assessed by annexin V staining and flow cytometry. Data are the means ± SEM of three independent experiments. **** p

    Journal: Cell Death and Differentiation

    Article Title: TRIM17 and TRIM28 antagonistically regulate the ubiquitination and anti-apoptotic activity of BCL2A1

    doi: 10.1038/s41418-018-0169-5

    Figure Lengend Snippet: TRIM17 invalidation induces BCL2A1 degradation and sensitizes melanoma cells to BRAF-targeted therapy. a Method outline depicting generation of TRIM17-depleted SK-MEL-28 cells by inducible CRISPR/Cas9 method and workflow of subsequent steps of analysis. b SK-MEL-28 cells expressing Cas9 and inducible sgRNAs against TRIM17 were treated for 72 h with 1 μg/ml doxycycline. Genomic DNA of cells was amplified by PCR, around the Cas9 cleavage sites targeted by the two sgTRIM17, and PCR products were analyzed by a T7 Endonuclease I assay. The presence of InDels was visualized by the digestion of the PCR products at the Cas9 cleavage sites by T7EI. c PLA was performed in SK-MEL-28 cells expressing DOX-inducible sgRNA against TRIM17, using anti-TRIM28 and anti-BCL2A1 antibodies. Note that the number of green bright spots indicating the very close proximity between endogenous TRIM28 and BCL2A1 proteins increases when expression of endogenous TRIM17 is inhibited by doxycycline treatment. Negative control was obtained by omitting the anti-BCL2A1 antibody. Scale bars, 10 μm. d The protein level of endogenous BCL2A1 was assessed by immunoblot using anti-BCL2A1 antibody in total protein extracts from non-transduced SK-MEL-28 cells left untreated or treated with 20 μM MG132 for 6 h, or from transduced cells expressing Cas9 alone or Cas9 together with inducible sgRNAs against BCL2A1 or mouse bim (negative control) and treated or not with 1 μg/ml doxycycline for 72 h, as indicated. e Two sgRNAs against TRIM17 were induced in SK-MEL-28 cells by doxycycline treatment for 72 h and protein level of endogenous BCL2A1 was assessed by immunoblot using anti-BCL2A1 antibody. f SK-MEL-28 cells expressing Cas9 and the indicated sgRNAs were treated with 1 μg/ml doxycycline for 72 h and subsequently treated with 20 μM PLX4720 (or DMSO control) for 48 h. PLX4720-specific induced apoptosis (SIA) was assessed by annexin V staining and flow cytometry. Data are the means ± SEM of three independent experiments. **** p

    Article Snippet: RNA preparation and real-time quantitative RT-PCR Total RNA was extracted using the RNAqueous® kit (Ambion) and treated with DNase I from the DNA-free™ kit (Ambion) according to manufacturer’s instructions.

    Techniques: CRISPR, Expressing, Amplification, Polymerase Chain Reaction, T7EI Assay, Proximity Ligation Assay, Negative Control, Staining, Flow Cytometry

    Comparison of the growth rate of H3N2 A/Guangdong/ST798/2008 strains carrying either 526R or 526K PB2 in mixed cultures. ( a ) MDCK cells or ( b ) A549 cells were infected at an MOI of 0.001 with a mixture (1:1) of wild-type (WT) (PB2-526R) and variant (PB2 R526K) A/Guangdong/ST798/2008 and sequentially passaged four times. At each passage viral RNA was isolated from cell culture supernatants at 48 h post infection, and the PB2 gene amplified by RT-PCR and sequenced. The genetic code for WT PB2-526R is AGA; variant PB2 526K is AAA. The DNA sequence chromatograms shown are from one of the three MDCK ( a ) and one of the four A549 ( b ) experiments in which 526R outgrew 526K. Experiments were repeated four times.

    Journal: Nature Communications

    Article Title: The K526R substitution in viral protein PB2 enhances the effects of E627K on influenza virus replication

    doi: 10.1038/ncomms6509

    Figure Lengend Snippet: Comparison of the growth rate of H3N2 A/Guangdong/ST798/2008 strains carrying either 526R or 526K PB2 in mixed cultures. ( a ) MDCK cells or ( b ) A549 cells were infected at an MOI of 0.001 with a mixture (1:1) of wild-type (WT) (PB2-526R) and variant (PB2 R526K) A/Guangdong/ST798/2008 and sequentially passaged four times. At each passage viral RNA was isolated from cell culture supernatants at 48 h post infection, and the PB2 gene amplified by RT-PCR and sequenced. The genetic code for WT PB2-526R is AGA; variant PB2 526K is AAA. The DNA sequence chromatograms shown are from one of the three MDCK ( a ) and one of the four A549 ( b ) experiments in which 526R outgrew 526K. Experiments were repeated four times.

    Article Snippet: Plasmid DNA present in the total RNA was eliminated by deoxyribonuclease (DNase) treatment (Ambion), in accordance with the user manual.

    Techniques: Infection, Variant Assay, Isolation, Cell Culture, Amplification, Reverse Transcription Polymerase Chain Reaction, Sequencing