dnaase i treatment  (Thermo Fisher)


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    Structured Review

    Thermo Fisher dnaase i treatment
    Dnaase I Treatment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnaase i treatment/product/Thermo Fisher
    Average 95 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    dnaase i treatment - by Bioz Stars, 2020-04
    95/100 stars

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    Centrifugation:

    Article Title: Oxidation of the Cyclic Ethers 1,4-Dioxane and Tetrahydrofuran by a Monooxygenase in Two Pseudonocardia Species
    Article Snippet: The precipitate was collected by centrifugation (30 min at 21,000 × g at 4°C), washed once with 70% ethanol, and resuspended in 100 μl of nuclease-free water. .. Following elution with 100 μl of RNase-free water, RNA was subjected to DNase I treatments using a DNA-free kit (Ambion, Carlsbad, CA), according to the manufacturer's instructions, until all of the contaminating DNA was removed (i.e., when qPCR analysis showed a threshold cycle [ CT ] value of > 35).

    Article Title: Serendipita indica E5′ NT modulates extracellular nucleotide levels in the plant apoplast and affects fungal colonization
    Article Snippet: Subsequently, 250 μl chloroform : isoamyl‐alcohol (24:1) was added followed by an additional incubation for 5 min at RT and centrifugation. .. Contaminating genomic DNA was removed by DNase I treatments (Thermo Fisher Scientific, USA) according to manufacturer's protocol.

    Amplification:

    Article Title: Non-essentiality of canonical cell division genes in the planctomycete Planctopirus limnophila
    Article Snippet: DNase I treatment was performed with a DNA-free kit (Ambion). .. The absence of contaminating DNA was then confirmed by PCR amplification.

    Synthesized:

    Article Title: Transcriptional response of Burkholderia cenocepacia J2315 sessile cells to treatments with high doses of hydrogen peroxide and sodium hypochlorite
    Article Snippet: .. Biofilm formation, treatments, RNA extraction and DNase I treatments were performed as described above. cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA) using 1 μg of total RNA as starting material. .. Subsequently, cDNA was diluted (1:10) in DEPC water (Invitrogen) and stored at -20°C until further use.

    Article Title: Serendipita indica E5′ NT modulates extracellular nucleotide levels in the plant apoplast and affects fungal colonization
    Article Snippet: Contaminating genomic DNA was removed by DNase I treatments (Thermo Fisher Scientific, USA) according to manufacturer's protocol. .. First‐strand cDNA was synthesized with 1 μg of total RNA primed with Oligo‐dT and random hexamer using First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA).

    Cytometry:

    Article Title: Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications
    Article Snippet: The nickase treatment was done as before , with GFP(CAG)101 cells transfected with either pPN10, an empty plasmid, or pPN10-gCTG together with a vector expressing the Cas9 D10A nickase variant using Lipofectamine 2000 (Life Technologies) on day 1 and again on day 5 and 8. .. GFP intensities were determined using an Accuri C6 flow cytometer from BD.

    Construct:

    Article Title: Developmental chromatin restriction of pro-growth gene networks acts as an epigenetic barrier to axon regeneration in cortical neurons
    Article Snippet: At 3DIV, total RNA was extracted from neurons in culture using TRIzol reagent (10296028-Thermofisher, Waltham, MA) according to manufacturer’s instructions followed by DNAase-I treatment (EN052-Thermofisher, Waltham, MA). .. 100 ng of total RNA from three separate cultures were used to construct replicate libraries for each timepoint.

    SYBR Green Assay:

    Article Title: Proteomic Delineation of the ArcA Regulon in Salmonella Typhimurium During Anaerobiosis
    Article Snippet: The extracted RNA samples were subjected to DNase I treatment using the turbo DNA Free Kit (Ambion) to remove any genomic DNA contaminants. .. Quantitative PCR was performed by using the SYBR Green PCR master mix (Applied Biosystems, Foster City, CA) and specific primers on a StepOnePlus Real-time PCR system (ABI).

    Microarray:

    Article Title: Transcriptional response of Burkholderia cenocepacia J2315 sessile cells to treatments with high doses of hydrogen peroxide and sodium hypochlorite
    Article Snippet: In addition to the confirmation of the microarray results for the 30 min treated biofilms, the expression of the selected genes was also examined in biofilms treated with H2 O2 for 15 and 60 min. Five biological replicates were included for each test condition. .. Biofilm formation, treatments, RNA extraction and DNase I treatments were performed as described above. cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA) using 1 μg of total RNA as starting material.

    Random Hexamer Labeling:

    Article Title: Proteomic Delineation of the ArcA Regulon in Salmonella Typhimurium During Anaerobiosis
    Article Snippet: The extracted RNA samples were subjected to DNase I treatment using the turbo DNA Free Kit (Ambion) to remove any genomic DNA contaminants. .. Reverse transcription was performed by using Super Script II reverse transcriptase (Invitrogen, Carlsbad, CA) and random hexamer (Invitrogen) to generate cDNA.

    Article Title: Serendipita indica E5′ NT modulates extracellular nucleotide levels in the plant apoplast and affects fungal colonization
    Article Snippet: Contaminating genomic DNA was removed by DNase I treatments (Thermo Fisher Scientific, USA) according to manufacturer's protocol. .. First‐strand cDNA was synthesized with 1 μg of total RNA primed with Oligo‐dT and random hexamer using First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA).

    Infection:

    Article Title: Modularity and evolutionary constraints in a baculovirus gene regulatory network
    Article Snippet: Paragraph title: Cell lines and infection kinetic: virus infection, RNA extraction and reverse transcription ... RNA samples were subjected to DNase I treatments (DNA-free, Ambion) according to the manufacturer’s protocols.

    Expressing:

    Article Title: Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications
    Article Snippet: .. The nickase treatment was done as before , with GFP(CAG)101 cells transfected with either pPN10, an empty plasmid, or pPN10-gCTG together with a vector expressing the Cas9 D10A nickase variant using Lipofectamine 2000 (Life Technologies) on day 1 and again on day 5 and 8. .. On day 12, we harvested the cells and found that those transfected with pPN10-gCTG used for the SP-PCR in Fig. had an increase of 2.6 fold in the brightest 1% of the cells compared to pPN10-transfected cells.

    Article Title: Transcriptional response of Burkholderia cenocepacia J2315 sessile cells to treatments with high doses of hydrogen peroxide and sodium hypochlorite
    Article Snippet: In addition to the confirmation of the microarray results for the 30 min treated biofilms, the expression of the selected genes was also examined in biofilms treated with H2 O2 for 15 and 60 min. Five biological replicates were included for each test condition. .. Biofilm formation, treatments, RNA extraction and DNase I treatments were performed as described above. cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA) using 1 μg of total RNA as starting material.

    Article Title: Proteomic Delineation of the ArcA Regulon in Salmonella Typhimurium During Anaerobiosis
    Article Snippet: RT-qPCR measurements of gene expression were performed as previously described ( ). .. The extracted RNA samples were subjected to DNase I treatment using the turbo DNA Free Kit (Ambion) to remove any genomic DNA contaminants.

    Article Title: Oxidation of the Cyclic Ethers 1,4-Dioxane and Tetrahydrofuran by a Monooxygenase in Two Pseudonocardia Species
    Article Snippet: Following elution with 100 μl of RNase-free water, RNA was subjected to DNase I treatments using a DNA-free kit (Ambion, Carlsbad, CA), according to the manufacturer's instructions, until all of the contaminating DNA was removed (i.e., when qPCR analysis showed a threshold cycle [ CT ] value of > 35). .. DNase-treated RNA was subjected to a final cleanup using an RNeasy kit before gene expression analyses were performed.

    Hybridization:

    Article Title: Characterization of the Effects of Salicylidene Acylhydrazide Compounds on Type III Secretion in Escherichia coli O157:H7 ▿ O157:H7 ▿ †
    Article Snippet: Contaminating DNA was removed by DNase I treatment (Ambion). .. The cDNA was hybridized to 70-mer spotted oligonucleotide arrays containing open reading frames from E. coli K-12, E. coli Sakai VT2, and E. coli EDL933 (University of Birmingham, United Kingdom) using a Genomic Solutions GeneTac hybridization machine.

    Transfection:

    Article Title: Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications
    Article Snippet: .. The nickase treatment was done as before , with GFP(CAG)101 cells transfected with either pPN10, an empty plasmid, or pPN10-gCTG together with a vector expressing the Cas9 D10A nickase variant using Lipofectamine 2000 (Life Technologies) on day 1 and again on day 5 and 8. .. On day 12, we harvested the cells and found that those transfected with pPN10-gCTG used for the SP-PCR in Fig. had an increase of 2.6 fold in the brightest 1% of the cells compared to pPN10-transfected cells.

    Article Title: Developmental chromatin restriction of pro-growth gene networks acts as an epigenetic barrier to axon regeneration in cortical neurons
    Article Snippet: Cells were transfected then plated at high density (300,000 cells/well) and maintained in culture for 2 days (P5) or 2 weeks (P14) at 37 °C, 5% CO2 in Enriched Neurobasal (ENB) media (10888-022-Thermofisher, Waltham, MA). .. At 3DIV, total RNA was extracted from neurons in culture using TRIzol reagent (10296028-Thermofisher, Waltham, MA) according to manufacturer’s instructions followed by DNAase-I treatment (EN052-Thermofisher, Waltham, MA).

    Cell Harvesting:

    Article Title: Oxidation of the Cyclic Ethers 1,4-Dioxane and Tetrahydrofuran by a Monooxygenase in Two Pseudonocardia Species
    Article Snippet: Paragraph title: Cell harvesting and RNA extraction for transcriptional studies. ... Following elution with 100 μl of RNase-free water, RNA was subjected to DNase I treatments using a DNA-free kit (Ambion, Carlsbad, CA), according to the manufacturer's instructions, until all of the contaminating DNA was removed (i.e., when qPCR analysis showed a threshold cycle [ CT ] value of > 35).

    Cell Culture:

    Article Title: Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications
    Article Snippet: Paragraph title: Cell culture ... The nickase treatment was done as before , with GFP(CAG)101 cells transfected with either pPN10, an empty plasmid, or pPN10-gCTG together with a vector expressing the Cas9 D10A nickase variant using Lipofectamine 2000 (Life Technologies) on day 1 and again on day 5 and 8.

    Article Title: Developmental chromatin restriction of pro-growth gene networks acts as an epigenetic barrier to axon regeneration in cortical neurons
    Article Snippet: Paragraph title: Cortical cell culture and RNA-seq ... At 3DIV, total RNA was extracted from neurons in culture using TRIzol reagent (10296028-Thermofisher, Waltham, MA) according to manufacturer’s instructions followed by DNAase-I treatment (EN052-Thermofisher, Waltham, MA).

    Polymerase Chain Reaction:

    Article Title: Proteomic Delineation of the ArcA Regulon in Salmonella Typhimurium During Anaerobiosis
    Article Snippet: Paragraph title: Reverse Transcription-Quantitative PCR (RT-qPCR) ... The extracted RNA samples were subjected to DNase I treatment using the turbo DNA Free Kit (Ambion) to remove any genomic DNA contaminants.

    Article Title: Non-essentiality of canonical cell division genes in the planctomycete Planctopirus limnophila
    Article Snippet: DNase I treatment was performed with a DNA-free kit (Ambion). .. The absence of contaminating DNA was then confirmed by PCR amplification.

    DNA Extraction:

    Article Title: Serendipita indica E5′ NT modulates extracellular nucleotide levels in the plant apoplast and affects fungal colonization
    Article Snippet: Real‐time PCR analysis DNA isolation was performed from ~200 mg of ground material. .. Contaminating genomic DNA was removed by DNase I treatments (Thermo Fisher Scientific, USA) according to manufacturer's protocol.

    RNA Sequencing Assay:

    Article Title: Developmental chromatin restriction of pro-growth gene networks acts as an epigenetic barrier to axon regeneration in cortical neurons
    Article Snippet: Paragraph title: Cortical cell culture and RNA-seq ... At 3DIV, total RNA was extracted from neurons in culture using TRIzol reagent (10296028-Thermofisher, Waltham, MA) according to manufacturer’s instructions followed by DNAase-I treatment (EN052-Thermofisher, Waltham, MA).

    RNA HS Assay:

    Article Title: Developmental chromatin restriction of pro-growth gene networks acts as an epigenetic barrier to axon regeneration in cortical neurons
    Article Snippet: At 3DIV, total RNA was extracted from neurons in culture using TRIzol reagent (10296028-Thermofisher, Waltham, MA) according to manufacturer’s instructions followed by DNAase-I treatment (EN052-Thermofisher, Waltham, MA). .. Total RNA quantification ( -RNA HS assay, Qubit Fluorometer-ThermoFisher, Waltham, MA ) and quality assessment (50671512-RNA nano chips, Bioanalyzer, Agilent, Santa Clara, CA) were performed according to guidelines recommended by ENCODE consortia (goo.gl/euW5t4).

    Isolation:

    Article Title: Serendipita indica E5′ NT modulates extracellular nucleotide levels in the plant apoplast and affects fungal colonization
    Article Snippet: Total RNA was isolated from 200 mg of ground fungal material or colonized and non‐colonized plant root material using the TRIzol® reagent (Invitrogen, USA) as described previously . .. Contaminating genomic DNA was removed by DNase I treatments (Thermo Fisher Scientific, USA) according to manufacturer's protocol.

    Article Title: Evaluation of Diabetes Effects on the Expression of Leukemia Inhibitory Factor and Vascular Endothelial Growth Factor A Genes and Proteins at the Time of Endometrial Receptivity after Superovulation in Rat Model
    Article Snippet: Paragraph title: Total RNA isolation and cDNA synthesis ... Genomic DNA was eliminated by DNase I treatment using DNase I set (Fermentas, Lithuania).

    Flow Cytometry:

    Article Title: Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications
    Article Snippet: The nickase treatment was done as before , with GFP(CAG)101 cells transfected with either pPN10, an empty plasmid, or pPN10-gCTG together with a vector expressing the Cas9 D10A nickase variant using Lipofectamine 2000 (Life Technologies) on day 1 and again on day 5 and 8. .. GFP intensities were determined using an Accuri C6 flow cytometer from BD.

    Labeling:

    Article Title: Characterization of the Effects of Salicylidene Acylhydrazide Compounds on Type III Secretion in Escherichia coli O157:H7 ▿ O157:H7 ▿ †
    Article Snippet: Contaminating DNA was removed by DNase I treatment (Ambion). .. Synthesis of cDNA and labeling of total RNA were performed using an Amersham CyScribe postlabeling kit according to the manufacturer's instructions.

    Purification:

    Article Title: Oxidation of the Cyclic Ethers 1,4-Dioxane and Tetrahydrofuran by a Monooxygenase in Two Pseudonocardia Species
    Article Snippet: To obtain RNA, resuspended nucleic acids were initially separated with an AllPrep kit (Qiagen, Valencia, CA), and then the RNA was purified using an RNeasy kit (Qiagen). .. Following elution with 100 μl of RNase-free water, RNA was subjected to DNase I treatments using a DNA-free kit (Ambion, Carlsbad, CA), according to the manufacturer's instructions, until all of the contaminating DNA was removed (i.e., when qPCR analysis showed a threshold cycle [ CT ] value of > 35).

    Article Title: Non-essentiality of canonical cell division genes in the planctomycete Planctopirus limnophila
    Article Snippet: DNase I treatment was performed with a DNA-free kit (Ambion). .. The samples were purified using RNAeasy columns (Quiagen) and RNA quality was confirmed by non-denaturing agarose gel electrophoresis.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Non-essentiality of canonical cell division genes in the planctomycete Planctopirus limnophila
    Article Snippet: Paragraph title: Semi-quantitative RT-PCR ... DNase I treatment was performed with a DNA-free kit (Ambion).

    Quantitative RT-PCR:

    Article Title: Transcriptional response of Burkholderia cenocepacia J2315 sessile cells to treatments with high doses of hydrogen peroxide and sodium hypochlorite
    Article Snippet: Paragraph title: Quantitative RT-PCR ... Biofilm formation, treatments, RNA extraction and DNase I treatments were performed as described above. cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA) using 1 μg of total RNA as starting material.

    Article Title: Proteomic Delineation of the ArcA Regulon in Salmonella Typhimurium During Anaerobiosis
    Article Snippet: Paragraph title: Reverse Transcription-Quantitative PCR (RT-qPCR) ... The extracted RNA samples were subjected to DNase I treatment using the turbo DNA Free Kit (Ambion) to remove any genomic DNA contaminants.

    Article Title: Serendipita indica E5′ NT modulates extracellular nucleotide levels in the plant apoplast and affects fungal colonization
    Article Snippet: Contaminating genomic DNA was removed by DNase I treatments (Thermo Fisher Scientific, USA) according to manufacturer's protocol. .. The resulting cDNA preparations were diluted to a final concentration of 2.5 ng/μl, and 4 μl of each cDNA sample was used for qRT–PCR.

    Plasmid Preparation:

    Article Title: Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications
    Article Snippet: .. The nickase treatment was done as before , with GFP(CAG)101 cells transfected with either pPN10, an empty plasmid, or pPN10-gCTG together with a vector expressing the Cas9 D10A nickase variant using Lipofectamine 2000 (Life Technologies) on day 1 and again on day 5 and 8. .. On day 12, we harvested the cells and found that those transfected with pPN10-gCTG used for the SP-PCR in Fig. had an increase of 2.6 fold in the brightest 1% of the cells compared to pPN10-transfected cells.

    Software:

    Article Title: Transcriptional response of Burkholderia cenocepacia J2315 sessile cells to treatments with high doses of hydrogen peroxide and sodium hypochlorite
    Article Snippet: Biofilm formation, treatments, RNA extraction and DNase I treatments were performed as described above. cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA) using 1 μg of total RNA as starting material. .. Forward and reverse primers were developed using Primer Express Software (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer's instructions and they were compared to the B. cenocepacia J2315 database using BLAST to determine their specificity.

    Real-time Polymerase Chain Reaction:

    Article Title: Transcriptional response of Burkholderia cenocepacia J2315 sessile cells to treatments with high doses of hydrogen peroxide and sodium hypochlorite
    Article Snippet: Biofilm formation, treatments, RNA extraction and DNase I treatments were performed as described above. cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA) using 1 μg of total RNA as starting material. .. All qPCR experiments were performed on a Bio-Rad CFX96 Real-Time System C1000 Thermal Cycler.

    Article Title: Proteomic Delineation of the ArcA Regulon in Salmonella Typhimurium During Anaerobiosis
    Article Snippet: The extracted RNA samples were subjected to DNase I treatment using the turbo DNA Free Kit (Ambion) to remove any genomic DNA contaminants. .. Quantitative PCR was performed by using the SYBR Green PCR master mix (Applied Biosystems, Foster City, CA) and specific primers on a StepOnePlus Real-time PCR system (ABI).

    Article Title: Oxidation of the Cyclic Ethers 1,4-Dioxane and Tetrahydrofuran by a Monooxygenase in Two Pseudonocardia Species
    Article Snippet: .. Following elution with 100 μl of RNase-free water, RNA was subjected to DNase I treatments using a DNA-free kit (Ambion, Carlsbad, CA), according to the manufacturer's instructions, until all of the contaminating DNA was removed (i.e., when qPCR analysis showed a threshold cycle [ CT ] value of > 35). .. DNase-treated RNA was subjected to a final cleanup using an RNeasy kit before gene expression analyses were performed.

    Article Title: Serendipita indica E5′ NT modulates extracellular nucleotide levels in the plant apoplast and affects fungal colonization
    Article Snippet: Paragraph title: Real‐time PCR analysis ... Contaminating genomic DNA was removed by DNase I treatments (Thermo Fisher Scientific, USA) according to manufacturer's protocol.

    Article Title: Modularity and evolutionary constraints in a baculovirus gene regulatory network
    Article Snippet: RNA samples were subjected to DNase I treatments (DNA-free, Ambion) according to the manufacturer’s protocols. .. The cDNAs were mixed and used in the real-time PCR reactions.

    RNA Extraction:

    Article Title: Transcriptional response of Burkholderia cenocepacia J2315 sessile cells to treatments with high doses of hydrogen peroxide and sodium hypochlorite
    Article Snippet: .. Biofilm formation, treatments, RNA extraction and DNase I treatments were performed as described above. cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA) using 1 μg of total RNA as starting material. .. Subsequently, cDNA was diluted (1:10) in DEPC water (Invitrogen) and stored at -20°C until further use.

    Article Title: Oxidation of the Cyclic Ethers 1,4-Dioxane and Tetrahydrofuran by a Monooxygenase in Two Pseudonocardia Species
    Article Snippet: Paragraph title: Cell harvesting and RNA extraction for transcriptional studies. ... Following elution with 100 μl of RNase-free water, RNA was subjected to DNase I treatments using a DNA-free kit (Ambion, Carlsbad, CA), according to the manufacturer's instructions, until all of the contaminating DNA was removed (i.e., when qPCR analysis showed a threshold cycle [ CT ] value of > 35).

    Article Title: Modularity and evolutionary constraints in a baculovirus gene regulatory network
    Article Snippet: Paragraph title: Cell lines and infection kinetic: virus infection, RNA extraction and reverse transcription ... RNA samples were subjected to DNase I treatments (DNA-free, Ambion) according to the manufacturer’s protocols.

    Article Title: Overexpression of a modified eIF4E regulates potato virus Y resistance at the transcriptional level in potato
    Article Snippet: Paragraph title: RNA extraction ... An additional DNase I treatment (Ambion) was included to remove contaminating genomic DNA.

    Article Title: Non-essentiality of canonical cell division genes in the planctomycete Planctopirus limnophila
    Article Snippet: Semi-quantitative RT-PCR Total RNA extraction was carried out as previously described from 20 ml of wild type cultures at exponential (OD600 ~0,4) and stationary phase (OD600 ~1,5). .. DNase I treatment was performed with a DNA-free kit (Ambion).

    Agarose Gel Electrophoresis:

    Article Title: Overexpression of a modified eIF4E regulates potato virus Y resistance at the transcriptional level in potato
    Article Snippet: An additional DNase I treatment (Ambion) was included to remove contaminating genomic DNA. .. Quality of the RNA was assessed by agarose gel electrophoresis.

    Article Title: Non-essentiality of canonical cell division genes in the planctomycete Planctopirus limnophila
    Article Snippet: DNase I treatment was performed with a DNA-free kit (Ambion). .. The samples were purified using RNAeasy columns (Quiagen) and RNA quality was confirmed by non-denaturing agarose gel electrophoresis.

    Article Title: Evaluation of Diabetes Effects on the Expression of Leukemia Inhibitory Factor and Vascular Endothelial Growth Factor A Genes and Proteins at the Time of Endometrial Receptivity after Superovulation in Rat Model
    Article Snippet: The integrity of the extracted RNA was verified by 1% agarose gel electrophoresis. .. Genomic DNA was eliminated by DNase I treatment using DNase I set (Fermentas, Lithuania).

    Incubation:

    Article Title: Oxidation of the Cyclic Ethers 1,4-Dioxane and Tetrahydrofuran by a Monooxygenase in Two Pseudonocardia Species
    Article Snippet: Nucleic acids were precipitated with 0.1 volume of 3 M sodium acetate and 1 volume of ice-cold isopropanol, followed by incubation at −20°C overnight. .. Following elution with 100 μl of RNase-free water, RNA was subjected to DNase I treatments using a DNA-free kit (Ambion, Carlsbad, CA), according to the manufacturer's instructions, until all of the contaminating DNA was removed (i.e., when qPCR analysis showed a threshold cycle [ CT ] value of > 35).

    Article Title: Serendipita indica E5′ NT modulates extracellular nucleotide levels in the plant apoplast and affects fungal colonization
    Article Snippet: Subsequently, 250 μl chloroform : isoamyl‐alcohol (24:1) was added followed by an additional incubation for 5 min at RT and centrifugation. .. Contaminating genomic DNA was removed by DNase I treatments (Thermo Fisher Scientific, USA) according to manufacturer's protocol.

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment
    Article Snippet: .. Enzyme treatment of C. jejuni biofilms For DNase I treatments, unless otherwise stated, a volume of 4 μl DNase I enzyme (Fermentas), giving a final concentration within the biofilm of 4 U/ml v/v and 4 μl of DNase I buffer (Fermentas) were added to each test tube, along with 1 ml of diluted cell suspension at either the start of the static incubation or after 12, 24, 36, or 48 h of static incubation. .. Following treatment, static cultures were placed back in 37°C, aerobic conditions to complete the 48 h incubation before staining with crystal violet to allow biofilm quantification.

    Article Title: Modularity and evolutionary constraints in a baculovirus gene regulatory network
    Article Snippet: Cultures of 105 UFL-AG-286 e IPLB-SF-9 cells were infected at a multiplicity of infection (MOI) of 10 and incubated at 28°C for 1h to allow infection synchronization. .. RNA samples were subjected to DNase I treatments (DNA-free, Ambion) according to the manufacturer’s protocols.

    Concentration Assay:

    Article Title: Characterization of the Effects of Salicylidene Acylhydrazide Compounds on Type III Secretion in Escherichia coli O157:H7 ▿ O157:H7 ▿ †
    Article Snippet: An inhibitor was added to test cultures at a final concentration of 20 μM, and an equal volume of DMSO was added to reference cultures. .. Contaminating DNA was removed by DNase I treatment (Ambion).

    Article Title: Serendipita indica E5′ NT modulates extracellular nucleotide levels in the plant apoplast and affects fungal colonization
    Article Snippet: Contaminating genomic DNA was removed by DNase I treatments (Thermo Fisher Scientific, USA) according to manufacturer's protocol. .. The resulting cDNA preparations were diluted to a final concentration of 2.5 ng/μl, and 4 μl of each cDNA sample was used for qRT–PCR.

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment
    Article Snippet: .. Enzyme treatment of C. jejuni biofilms For DNase I treatments, unless otherwise stated, a volume of 4 μl DNase I enzyme (Fermentas), giving a final concentration within the biofilm of 4 U/ml v/v and 4 μl of DNase I buffer (Fermentas) were added to each test tube, along with 1 ml of diluted cell suspension at either the start of the static incubation or after 12, 24, 36, or 48 h of static incubation. .. Following treatment, static cultures were placed back in 37°C, aerobic conditions to complete the 48 h incubation before staining with crystal violet to allow biofilm quantification.

    Article Title: Evaluation of Diabetes Effects on the Expression of Leukemia Inhibitory Factor and Vascular Endothelial Growth Factor A Genes and Proteins at the Time of Endometrial Receptivity after Superovulation in Rat Model
    Article Snippet: The concentration of total RNA was measured by NanoDrop instrument (Nanolytik, Germany) for each sample at 260 nm OD (Optical density), and they were stored at −80°C for further analysis. .. Genomic DNA was eliminated by DNase I treatment using DNase I set (Fermentas, Lithuania).

    Staining:

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment
    Article Snippet: Enzyme treatment of C. jejuni biofilms For DNase I treatments, unless otherwise stated, a volume of 4 μl DNase I enzyme (Fermentas), giving a final concentration within the biofilm of 4 U/ml v/v and 4 μl of DNase I buffer (Fermentas) were added to each test tube, along with 1 ml of diluted cell suspension at either the start of the static incubation or after 12, 24, 36, or 48 h of static incubation. .. Following treatment, static cultures were placed back in 37°C, aerobic conditions to complete the 48 h incubation before staining with crystal violet to allow biofilm quantification.

    Variant Assay:

    Article Title: Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications
    Article Snippet: .. The nickase treatment was done as before , with GFP(CAG)101 cells transfected with either pPN10, an empty plasmid, or pPN10-gCTG together with a vector expressing the Cas9 D10A nickase variant using Lipofectamine 2000 (Life Technologies) on day 1 and again on day 5 and 8. .. On day 12, we harvested the cells and found that those transfected with pPN10-gCTG used for the SP-PCR in Fig. had an increase of 2.6 fold in the brightest 1% of the cells compared to pPN10-transfected cells.

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  • 99
    Thermo Fisher dna digestion mix
    Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary <t>DNA</t> (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease <t>(DNase</t> I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)
    Dna Digestion Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna digestion mix/product/Thermo Fisher
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    dna digestion mix - by Bioz Stars, 2020-04
    99/100 stars
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    99
    Thermo Fisher dnase i treatment
    IR induces RIG-I binding to endogenous double-stranded RNAs A. HEK293 reporter cells were irradiated after transfection with either an empty vector, a full length human RIG-I, a RIG-I lacking CARD domains (RIG-I helicase/CTD), or a RIG-I harboring K858A and K861A mutations in the C-terminal domain (RIG-I K858A-K861A), in addition to an IFN-beta promoter-driven luciferase construct. A Renilla reporter construct served as a transfection control. Data are presented as mean fold-change relative to the non-irradiated empty vector control. B. Donor HEK293 cells were either unirradiated or treated with IR (3 or 6 Gy). Total RNA was purified and transferred to independent batches of HEK293 reporter cells transfected by RIG-I constructs as described in (A). A synthetic double-stranded RNA construct comprised of 5′-triphosphorylated dsRNA and an unphosphorylated counterpart served as positive and negative controls, respectively (inset). C. Experimental design for isolation and purification of RNA bound to RIG-I after exposure to IR. *To validate RNA sequencing data by qPCR experiments, UV crosslinking was performed prior to cell lysis and immunoprecipitation of RIG-I. See methods for further details. D. Purified RNA from total cellular extracts (Lanes 2 and 3) and complexes with RIG-I (Lanes 4 and 5). Lane 1 is the marker. Data are representative of at least 3 independent experiments. E. HEK293 cells over-expressing the HA-tagged full length RIG-I (Lanes 2 and 3), the RIG-I helicase-CTD mutant (Lanes 4 and 5) and the RIG-I K858A-K861A CTD mutant (Lanes 6 and 7) were either un-irradiated or exposed to IR (6 Gy), lysed and incubated with anti-HA monoclonal antibody to pulldown the respective WT and mutant RIG-I proteins. RIG-I diagrams illustrate the mechanism of RIG-I activation (adapted from [ 57 ]). In the inactive/unbound conformation, the CARD domain of RIG-I is folded to block the helicase domain from RNA binding RNA, but allows the CTD to search for its ligand. Upon binding of the blunt end of a dsRNA molecule to the CTD, the CARD domain opens to allow the helicase domain to bind the remaining dsRNA molecule. Absence of the CARD domain in the helicase/CTD mutant enables higher affinity binding to dsRNA ligands as compared to the full length RIG-I. The lysine residues at amino acid positions 858 and 861 have previously demonstrated importance in latching onto the 5′-triphosphorylated end of viral dsRNA ligands. F. RNA bound to RIG-I after exposure to IR (6 Gy) was treated with: RNase A (lane 3), dsRNA-specific RNase III (lane 4), single-strand specific nuclease S1 (lane 5) and <t>DNase</t> I (lane 7). Lane 2 shows the input and lanes 1 and 6 display markers.
    Dnase I Treatment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 295 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dnase treatment
    Colicin D-based molecular surgery and preparation of Lys3-modivariants. ( A ) Treatment of native tRNA Lys 3 (gray line, modifications indicated as gray dots) with Colicin D generated two fragments with 38 nt length (see PAGE analysis). Differences in migration distance were caused by 2′-3′-cyclic phosphates at the 3′ end of the 5′ fragment. ( B ) Due to similar fragment lengths, a DNA oligonucleotide (darker gray) was hybridized to the 3′ fragment. The corresponding band shift of the double-stranded <t>RNA/DNA</t> hybrid enabled separation and isolation of both tRNA fragments. RNAs were then treated with DNaseI and FastAP. ( C ) Prephosphorylated 5′ and 3′ fragments were subsequently annealed together with unmodified RNAs (black lines) on splint DNA (indicated by the darker gray line, fluorescence marker displayed as a star) which contains a 3′ fluorescein residue. Ligation and <t>DNase</t> treatment yielded mv#1 and mv#2 as shown in the respective PAGE analysis. Evaluation of the fluorescence channel revealed successful digestion of DNA splint in modivariant preparations.
    Dnase Treatment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 753 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Journal: Nature Communications

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

    doi: 10.1038/s41467-018-02866-0

    Figure Lengend Snippet: Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Article Snippet: To eliminate genomic DNA contamination, 1 μL of genomic DNA digestion mix (0.5× PrimeScript Buffer, 0.2 U of DNase I Amplification Grade, 1: 5 000 000 ERCC RNA Spike-In Mix I (Thermo Fisher) in RNase-free water) was added to 1 μL of the denatured sample.

    Techniques: Synthesized, Activity Assay, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Whisker Assay

    IR induces RIG-I binding to endogenous double-stranded RNAs A. HEK293 reporter cells were irradiated after transfection with either an empty vector, a full length human RIG-I, a RIG-I lacking CARD domains (RIG-I helicase/CTD), or a RIG-I harboring K858A and K861A mutations in the C-terminal domain (RIG-I K858A-K861A), in addition to an IFN-beta promoter-driven luciferase construct. A Renilla reporter construct served as a transfection control. Data are presented as mean fold-change relative to the non-irradiated empty vector control. B. Donor HEK293 cells were either unirradiated or treated with IR (3 or 6 Gy). Total RNA was purified and transferred to independent batches of HEK293 reporter cells transfected by RIG-I constructs as described in (A). A synthetic double-stranded RNA construct comprised of 5′-triphosphorylated dsRNA and an unphosphorylated counterpart served as positive and negative controls, respectively (inset). C. Experimental design for isolation and purification of RNA bound to RIG-I after exposure to IR. *To validate RNA sequencing data by qPCR experiments, UV crosslinking was performed prior to cell lysis and immunoprecipitation of RIG-I. See methods for further details. D. Purified RNA from total cellular extracts (Lanes 2 and 3) and complexes with RIG-I (Lanes 4 and 5). Lane 1 is the marker. Data are representative of at least 3 independent experiments. E. HEK293 cells over-expressing the HA-tagged full length RIG-I (Lanes 2 and 3), the RIG-I helicase-CTD mutant (Lanes 4 and 5) and the RIG-I K858A-K861A CTD mutant (Lanes 6 and 7) were either un-irradiated or exposed to IR (6 Gy), lysed and incubated with anti-HA monoclonal antibody to pulldown the respective WT and mutant RIG-I proteins. RIG-I diagrams illustrate the mechanism of RIG-I activation (adapted from [ 57 ]). In the inactive/unbound conformation, the CARD domain of RIG-I is folded to block the helicase domain from RNA binding RNA, but allows the CTD to search for its ligand. Upon binding of the blunt end of a dsRNA molecule to the CTD, the CARD domain opens to allow the helicase domain to bind the remaining dsRNA molecule. Absence of the CARD domain in the helicase/CTD mutant enables higher affinity binding to dsRNA ligands as compared to the full length RIG-I. The lysine residues at amino acid positions 858 and 861 have previously demonstrated importance in latching onto the 5′-triphosphorylated end of viral dsRNA ligands. F. RNA bound to RIG-I after exposure to IR (6 Gy) was treated with: RNase A (lane 3), dsRNA-specific RNase III (lane 4), single-strand specific nuclease S1 (lane 5) and DNase I (lane 7). Lane 2 shows the input and lanes 1 and 6 display markers.

    Journal: Oncotarget

    Article Title: Cancer therapies activate RIG-I-like receptor pathway through endogenous non-coding RNAs

    doi: 10.18632/oncotarget.8420

    Figure Lengend Snippet: IR induces RIG-I binding to endogenous double-stranded RNAs A. HEK293 reporter cells were irradiated after transfection with either an empty vector, a full length human RIG-I, a RIG-I lacking CARD domains (RIG-I helicase/CTD), or a RIG-I harboring K858A and K861A mutations in the C-terminal domain (RIG-I K858A-K861A), in addition to an IFN-beta promoter-driven luciferase construct. A Renilla reporter construct served as a transfection control. Data are presented as mean fold-change relative to the non-irradiated empty vector control. B. Donor HEK293 cells were either unirradiated or treated with IR (3 or 6 Gy). Total RNA was purified and transferred to independent batches of HEK293 reporter cells transfected by RIG-I constructs as described in (A). A synthetic double-stranded RNA construct comprised of 5′-triphosphorylated dsRNA and an unphosphorylated counterpart served as positive and negative controls, respectively (inset). C. Experimental design for isolation and purification of RNA bound to RIG-I after exposure to IR. *To validate RNA sequencing data by qPCR experiments, UV crosslinking was performed prior to cell lysis and immunoprecipitation of RIG-I. See methods for further details. D. Purified RNA from total cellular extracts (Lanes 2 and 3) and complexes with RIG-I (Lanes 4 and 5). Lane 1 is the marker. Data are representative of at least 3 independent experiments. E. HEK293 cells over-expressing the HA-tagged full length RIG-I (Lanes 2 and 3), the RIG-I helicase-CTD mutant (Lanes 4 and 5) and the RIG-I K858A-K861A CTD mutant (Lanes 6 and 7) were either un-irradiated or exposed to IR (6 Gy), lysed and incubated with anti-HA monoclonal antibody to pulldown the respective WT and mutant RIG-I proteins. RIG-I diagrams illustrate the mechanism of RIG-I activation (adapted from [ 57 ]). In the inactive/unbound conformation, the CARD domain of RIG-I is folded to block the helicase domain from RNA binding RNA, but allows the CTD to search for its ligand. Upon binding of the blunt end of a dsRNA molecule to the CTD, the CARD domain opens to allow the helicase domain to bind the remaining dsRNA molecule. Absence of the CARD domain in the helicase/CTD mutant enables higher affinity binding to dsRNA ligands as compared to the full length RIG-I. The lysine residues at amino acid positions 858 and 861 have previously demonstrated importance in latching onto the 5′-triphosphorylated end of viral dsRNA ligands. F. RNA bound to RIG-I after exposure to IR (6 Gy) was treated with: RNase A (lane 3), dsRNA-specific RNase III (lane 4), single-strand specific nuclease S1 (lane 5) and DNase I (lane 7). Lane 2 shows the input and lanes 1 and 6 display markers.

    Article Snippet: qRT-PCR analysis 1 μg total RNA was subjected to DNase I treatment in a 30 μL reaction volume using DNase I, RNase-free (Thermo Scientific) following the manufacturer's protocol. cDNA was synthesized from 10 μL of the DNase treated RNA using the High-Capacity cDNA Reverse Transcription Kit (LifeTechnologies) following the manufacturer's protocol.

    Techniques: Binding Assay, Irradiation, Transfection, Plasmid Preparation, Luciferase, Construct, Purification, Isolation, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Lysis, Immunoprecipitation, Marker, Expressing, Mutagenesis, Incubation, Activation Assay, Blocking Assay, RNA Binding Assay

    BKPyV VP4 is not required for viral progeny release of the WW strain, but the VP4 start codon substitution M229I affects infectivity. (A) BKPyV load in DNase I-treated supernatants from RPTECs at 3 and 6 dpt, determined by real-time quantitative PCR and

    Journal: Journal of Virology

    Article Title: The Presumed Polyomavirus Viroporin VP4 of Simian Virus 40 or Human BK Polyomavirus Is Not Required for Viral Progeny Release

    doi: 10.1128/JVI.01326-16

    Figure Lengend Snippet: BKPyV VP4 is not required for viral progeny release of the WW strain, but the VP4 start codon substitution M229I affects infectivity. (A) BKPyV load in DNase I-treated supernatants from RPTECs at 3 and 6 dpt, determined by real-time quantitative PCR and

    Article Snippet: The DNase I treatment was performed directly on the supernatants using the Turbo DNA-free kit (AM1907; Thermo Fisher Scientific) according to the manufacturer's instructions.

    Techniques: Infection, Real-time Polymerase Chain Reaction

    SV40 VP4 is not required for viral progeny release, but the VP4 start codon substitution affects infectivity. (A) SV40 load in DNase I-treated supernatants from BS-C-1 cells and CV-1 cells at 1, 2, 4, and 5 dpt, determined by real-time quantitative PCR

    Journal: Journal of Virology

    Article Title: The Presumed Polyomavirus Viroporin VP4 of Simian Virus 40 or Human BK Polyomavirus Is Not Required for Viral Progeny Release

    doi: 10.1128/JVI.01326-16

    Figure Lengend Snippet: SV40 VP4 is not required for viral progeny release, but the VP4 start codon substitution affects infectivity. (A) SV40 load in DNase I-treated supernatants from BS-C-1 cells and CV-1 cells at 1, 2, 4, and 5 dpt, determined by real-time quantitative PCR

    Article Snippet: The DNase I treatment was performed directly on the supernatants using the Turbo DNA-free kit (AM1907; Thermo Fisher Scientific) according to the manufacturer's instructions.

    Techniques: Infection, Real-time Polymerase Chain Reaction

    Colicin D-based molecular surgery and preparation of Lys3-modivariants. ( A ) Treatment of native tRNA Lys 3 (gray line, modifications indicated as gray dots) with Colicin D generated two fragments with 38 nt length (see PAGE analysis). Differences in migration distance were caused by 2′-3′-cyclic phosphates at the 3′ end of the 5′ fragment. ( B ) Due to similar fragment lengths, a DNA oligonucleotide (darker gray) was hybridized to the 3′ fragment. The corresponding band shift of the double-stranded RNA/DNA hybrid enabled separation and isolation of both tRNA fragments. RNAs were then treated with DNaseI and FastAP. ( C ) Prephosphorylated 5′ and 3′ fragments were subsequently annealed together with unmodified RNAs (black lines) on splint DNA (indicated by the darker gray line, fluorescence marker displayed as a star) which contains a 3′ fluorescein residue. Ligation and DNase treatment yielded mv#1 and mv#2 as shown in the respective PAGE analysis. Evaluation of the fluorescence channel revealed successful digestion of DNA splint in modivariant preparations.

    Journal: Nucleic Acids Research

    Article Title: Double methylation of tRNA-U54 to 2′-O-methylthymidine (Tm) synergistically decreases immune response by Toll-like receptor 7

    doi: 10.1093/nar/gky644

    Figure Lengend Snippet: Colicin D-based molecular surgery and preparation of Lys3-modivariants. ( A ) Treatment of native tRNA Lys 3 (gray line, modifications indicated as gray dots) with Colicin D generated two fragments with 38 nt length (see PAGE analysis). Differences in migration distance were caused by 2′-3′-cyclic phosphates at the 3′ end of the 5′ fragment. ( B ) Due to similar fragment lengths, a DNA oligonucleotide (darker gray) was hybridized to the 3′ fragment. The corresponding band shift of the double-stranded RNA/DNA hybrid enabled separation and isolation of both tRNA fragments. RNAs were then treated with DNaseI and FastAP. ( C ) Prephosphorylated 5′ and 3′ fragments were subsequently annealed together with unmodified RNAs (black lines) on splint DNA (indicated by the darker gray line, fluorescence marker displayed as a star) which contains a 3′ fluorescein residue. Ligation and DNase treatment yielded mv#1 and mv#2 as shown in the respective PAGE analysis. Evaluation of the fluorescence channel revealed successful digestion of DNA splint in modivariant preparations.

    Article Snippet: DNase treatment and purification of RNA fragments To obtain the purified, dephosphorylated native fragment, the DNA/RNA hybrid solution was incubated with 0.1 U/μl FastAP (Thermo Scientific) and 1.25 U/μl DNaseI (Thermo Scientific) for 1 h at 37°C.

    Techniques: Generated, Polyacrylamide Gel Electrophoresis, Migration, Electrophoretic Mobility Shift Assay, Isolation, Fluorescence, Marker, Ligation