dna polymerase dnase i  (Thermo Fisher)


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    Name:
    DNA Polymerase I DNase I
    Description:
    DNA Polymerase I DNase I is an optimized mixture of both enzymes for efficient nick translation of DNA Application Labeling DNA with either radiolabeled or biotinylated nucleotides Source DNase I is purified from bovine pancreas DNA Polymerase I from E coli λ lysogen NM 964 Performance and Quality Testing Performance tested in nick translation reaction Unit Definition One unit of DNA Polymerase I in the absence of DNase I incorporates 10 nmol of total deoxyribonucleotide into acid precipitable material in 30 min at 37°C using a template•primer Unit Reaction Conditions 50 mM potassium phosphate pH 7 5 6 7 mM MgCl2 1 mM 2 mercaptoethanol 80 µg ml template•primer 32 µM dTTP 69 nM 3H dTTP and enzyme in 100 µl for 30 min at 37°C
    Catalog Number:
    18162016
    Price:
    None
    Applications:
    Cloning|Restriction Enzyme Cloning
    Category:
    Proteins Enzymes Peptides
    Buy from Supplier


    Structured Review

    Thermo Fisher dna polymerase dnase i
    DNA Polymerase I DNase I is an optimized mixture of both enzymes for efficient nick translation of DNA Application Labeling DNA with either radiolabeled or biotinylated nucleotides Source DNase I is purified from bovine pancreas DNA Polymerase I from E coli λ lysogen NM 964 Performance and Quality Testing Performance tested in nick translation reaction Unit Definition One unit of DNA Polymerase I in the absence of DNase I incorporates 10 nmol of total deoxyribonucleotide into acid precipitable material in 30 min at 37°C using a template•primer Unit Reaction Conditions 50 mM potassium phosphate pH 7 5 6 7 mM MgCl2 1 mM 2 mercaptoethanol 80 µg ml template•primer 32 µM dTTP 69 nM 3H dTTP and enzyme in 100 µl for 30 min at 37°C
    https://www.bioz.com/result/dna polymerase dnase i/product/Thermo Fisher
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dna polymerase dnase i - by Bioz Stars, 2020-05
    85/100 stars

    Related Products / Commonly Used Together

    dna probes
    green dutp
    α‐32 p
    red dutp

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    Related Articles

    Clone Assay:

    Article Title: Single copies of mutant KRAS and mutant PIK3CA cooperate in immortalized human epithelial cells to induce tumor formation
    Article Snippet: .. For probes, DNA was extracted from BAC clones and labeled by nick translation with Green dUTP or Red dUTP (Abbott Laboratories) using DNA polymerase/DNase I (Invitrogen). ..

    Amplification:

    Article Title: Tyramine biosynthesis in Enterococcus durans is transcriptionally regulated by the extracellular pH and tyrosine concentration
    Article Snippet: .. Double‐stranded DNA probes, amplified using the primers shown in , were radiolabelled with [α‐32 P]‐dATP by nick translation with DNA polymerase/DNase I (Invitrogen). .. Reverse transcription‐PCR RNA samples (2 µg of total RNA) were treated with 2 U of DNase (Fermentas), as described by the manufacturer, to eliminate any DNA contamination. cDNA was then synthesized from total RNA using the iScript™ cDNA Synthesis kit (Bio‐Rad) following the manufacturer's recommendations.

    Synthesized:

    Article Title: Identification of Xenopus CENP-A and an Associated Centromeric DNA Repeat D⃞
    Article Snippet: .. Digoxigenin-labeled probes were synthesized by nick translation of an ∼1-kb fragment of Fcr1 repeats, by using an optimized DNA polymerase I/DNase I mixture (Invitrogen) and digoxigenin-11-dUTP (Roche Diagnostics, Indianapolis, IN). .. Probes were detected with rhodamine-α-digoxigenin antibodies (Roche Diagnostics).

    Isolation:

    Article Title: A novel application of gene arrays: Escherichia coli array provides insight into the biology of the obligate endosymbiont of tsetse flies
    Article Snippet: .. Bacterial DNA isolated according to standard protocols was labeled with 32 P-dGTP or 32 P-dGTP and 32 PdTTP by nick translation using DNA Pol I/Dnase I (Nick translation system from GIBCO/BRL). .. Hybridization conditions were changed from 42°C to 45°C, in the presence of buffers containing 45% formamide, 5× Denhardt's (0.02% polyvinylpyrrolidone/0.02% Ficoll/0.02% BSA), 5× SSC, and 0.5% SDS.

    Nick Translation:

    Article Title: Genome Size Determination and Coding Capacity of Sodalis glossinidius, an Enteric Symbiont of Tsetse Flies, as Revealed by Hybridization to Escherichia coli Gene Arrays
    Article Snippet: .. DNA was radioactively labeled with [α-33 P]ATP by using a polymerase I/DNase I nick translation kit (GIBCO catalog no. 18160-010). ..

    Article Title: Single copies of mutant KRAS and mutant PIK3CA cooperate in immortalized human epithelial cells to induce tumor formation
    Article Snippet: .. For probes, DNA was extracted from BAC clones and labeled by nick translation with Green dUTP or Red dUTP (Abbott Laboratories) using DNA polymerase/DNase I (Invitrogen). ..

    Article Title: Identification of Xenopus CENP-A and an Associated Centromeric DNA Repeat D⃞
    Article Snippet: .. Probes were constructed by nick translation of an ∼1-kb fragment of Fcr1 repeats by using an optimized DNA polymerase I/DNase I mixture (Invitrogen). .. Southern hybridization was performed in Rapid-hyb buffer (Amersham Biosciences) according to the manufacturer's instructions.

    Article Title: A novel application of gene arrays: Escherichia coli array provides insight into the biology of the obligate endosymbiont of tsetse flies
    Article Snippet: .. Bacterial DNA isolated according to standard protocols was labeled with 32 P-dGTP or 32 P-dGTP and 32 PdTTP by nick translation using DNA Pol I/Dnase I (Nick translation system from GIBCO/BRL). .. Hybridization conditions were changed from 42°C to 45°C, in the presence of buffers containing 45% formamide, 5× Denhardt's (0.02% polyvinylpyrrolidone/0.02% Ficoll/0.02% BSA), 5× SSC, and 0.5% SDS.

    Article Title: Tyramine biosynthesis in Enterococcus durans is transcriptionally regulated by the extracellular pH and tyrosine concentration
    Article Snippet: .. Double‐stranded DNA probes, amplified using the primers shown in , were radiolabelled with [α‐32 P]‐dATP by nick translation with DNA polymerase/DNase I (Invitrogen). .. Reverse transcription‐PCR RNA samples (2 µg of total RNA) were treated with 2 U of DNase (Fermentas), as described by the manufacturer, to eliminate any DNA contamination. cDNA was then synthesized from total RNA using the iScript™ cDNA Synthesis kit (Bio‐Rad) following the manufacturer's recommendations.

    Article Title: Identification of Xenopus CENP-A and an Associated Centromeric DNA Repeat D⃞
    Article Snippet: .. Digoxigenin-labeled probes were synthesized by nick translation of an ∼1-kb fragment of Fcr1 repeats, by using an optimized DNA polymerase I/DNase I mixture (Invitrogen) and digoxigenin-11-dUTP (Roche Diagnostics, Indianapolis, IN). .. Probes were detected with rhodamine-α-digoxigenin antibodies (Roche Diagnostics).

    Article Title: Close, stable homolog juxtaposition during meiosis in budding yeast is dependent on meiotic recombination, occurs independently of synapsis, and is distinct from DSB-independent pairing contacts
    Article Snippet: .. Probe g was then reacted with Cy3-NHS ester dye (Amersham, catalog no. PA23001) per the manufacturer's instructions except that ∼5 μg of DNA was added to the dye reaction and incubated at 37°C for 2 h before quenching with 37.5 mM glycine (pH 8), followed by a further incubation at 37°C for 10 min. Probe q was labeled in the presence of 40 μM Alexa 488-dUTP (Molecular Probes) by nick translation using DNA Polymerase I/DNaseI (GIBCO BRL) in a reaction containing 50 mM Tris-HCl (pH 7.8), 5 mM MgCl2 , 10 mM 2-mercaptoethanol, 10 μg/mL BSA, 40 μM dATP, dCTP, and dGTP, 8 μM dTTP with an additional 4 U/μL DNase (GIBCO) at 15°C for 1 h. Reactions were stopped by the addition of EDTA (pH 8.0) to a final concentration of 50 mM and heated to 65°C for 15 min. Each probe was purified through a G-50 Sephadex spin column prior to slide hybridization. .. Hybridization and washing of probes was carried out essentially as described in except that 0.4× SSC, 1% BSA, and 4% dextran sulfate were used for the hybridization and no detection antibodies were used.

    Construct:

    Article Title: Identification of Xenopus CENP-A and an Associated Centromeric DNA Repeat D⃞
    Article Snippet: .. Probes were constructed by nick translation of an ∼1-kb fragment of Fcr1 repeats by using an optimized DNA polymerase I/DNase I mixture (Invitrogen). .. Southern hybridization was performed in Rapid-hyb buffer (Amersham Biosciences) according to the manufacturer's instructions.

    Purification:

    Article Title: Close, stable homolog juxtaposition during meiosis in budding yeast is dependent on meiotic recombination, occurs independently of synapsis, and is distinct from DSB-independent pairing contacts
    Article Snippet: .. Probe g was then reacted with Cy3-NHS ester dye (Amersham, catalog no. PA23001) per the manufacturer's instructions except that ∼5 μg of DNA was added to the dye reaction and incubated at 37°C for 2 h before quenching with 37.5 mM glycine (pH 8), followed by a further incubation at 37°C for 10 min. Probe q was labeled in the presence of 40 μM Alexa 488-dUTP (Molecular Probes) by nick translation using DNA Polymerase I/DNaseI (GIBCO BRL) in a reaction containing 50 mM Tris-HCl (pH 7.8), 5 mM MgCl2 , 10 mM 2-mercaptoethanol, 10 μg/mL BSA, 40 μM dATP, dCTP, and dGTP, 8 μM dTTP with an additional 4 U/μL DNase (GIBCO) at 15°C for 1 h. Reactions were stopped by the addition of EDTA (pH 8.0) to a final concentration of 50 mM and heated to 65°C for 15 min. Each probe was purified through a G-50 Sephadex spin column prior to slide hybridization. .. Hybridization and washing of probes was carried out essentially as described in except that 0.4× SSC, 1% BSA, and 4% dextran sulfate were used for the hybridization and no detection antibodies were used.

    Concentration Assay:

    Article Title: Close, stable homolog juxtaposition during meiosis in budding yeast is dependent on meiotic recombination, occurs independently of synapsis, and is distinct from DSB-independent pairing contacts
    Article Snippet: .. Probe g was then reacted with Cy3-NHS ester dye (Amersham, catalog no. PA23001) per the manufacturer's instructions except that ∼5 μg of DNA was added to the dye reaction and incubated at 37°C for 2 h before quenching with 37.5 mM glycine (pH 8), followed by a further incubation at 37°C for 10 min. Probe q was labeled in the presence of 40 μM Alexa 488-dUTP (Molecular Probes) by nick translation using DNA Polymerase I/DNaseI (GIBCO BRL) in a reaction containing 50 mM Tris-HCl (pH 7.8), 5 mM MgCl2 , 10 mM 2-mercaptoethanol, 10 μg/mL BSA, 40 μM dATP, dCTP, and dGTP, 8 μM dTTP with an additional 4 U/μL DNase (GIBCO) at 15°C for 1 h. Reactions were stopped by the addition of EDTA (pH 8.0) to a final concentration of 50 mM and heated to 65°C for 15 min. Each probe was purified through a G-50 Sephadex spin column prior to slide hybridization. .. Hybridization and washing of probes was carried out essentially as described in except that 0.4× SSC, 1% BSA, and 4% dextran sulfate were used for the hybridization and no detection antibodies were used.

    Incubation:

    Article Title: Close, stable homolog juxtaposition during meiosis in budding yeast is dependent on meiotic recombination, occurs independently of synapsis, and is distinct from DSB-independent pairing contacts
    Article Snippet: .. Probe g was then reacted with Cy3-NHS ester dye (Amersham, catalog no. PA23001) per the manufacturer's instructions except that ∼5 μg of DNA was added to the dye reaction and incubated at 37°C for 2 h before quenching with 37.5 mM glycine (pH 8), followed by a further incubation at 37°C for 10 min. Probe q was labeled in the presence of 40 μM Alexa 488-dUTP (Molecular Probes) by nick translation using DNA Polymerase I/DNaseI (GIBCO BRL) in a reaction containing 50 mM Tris-HCl (pH 7.8), 5 mM MgCl2 , 10 mM 2-mercaptoethanol, 10 μg/mL BSA, 40 μM dATP, dCTP, and dGTP, 8 μM dTTP with an additional 4 U/μL DNase (GIBCO) at 15°C for 1 h. Reactions were stopped by the addition of EDTA (pH 8.0) to a final concentration of 50 mM and heated to 65°C for 15 min. Each probe was purified through a G-50 Sephadex spin column prior to slide hybridization. .. Hybridization and washing of probes was carried out essentially as described in except that 0.4× SSC, 1% BSA, and 4% dextran sulfate were used for the hybridization and no detection antibodies were used.

    Labeling:

    Article Title: Genome Size Determination and Coding Capacity of Sodalis glossinidius, an Enteric Symbiont of Tsetse Flies, as Revealed by Hybridization to Escherichia coli Gene Arrays
    Article Snippet: .. DNA was radioactively labeled with [α-33 P]ATP by using a polymerase I/DNase I nick translation kit (GIBCO catalog no. 18160-010). ..

    Article Title: Single copies of mutant KRAS and mutant PIK3CA cooperate in immortalized human epithelial cells to induce tumor formation
    Article Snippet: .. For probes, DNA was extracted from BAC clones and labeled by nick translation with Green dUTP or Red dUTP (Abbott Laboratories) using DNA polymerase/DNase I (Invitrogen). ..

    Article Title: A novel application of gene arrays: Escherichia coli array provides insight into the biology of the obligate endosymbiont of tsetse flies
    Article Snippet: .. Bacterial DNA isolated according to standard protocols was labeled with 32 P-dGTP or 32 P-dGTP and 32 PdTTP by nick translation using DNA Pol I/Dnase I (Nick translation system from GIBCO/BRL). .. Hybridization conditions were changed from 42°C to 45°C, in the presence of buffers containing 45% formamide, 5× Denhardt's (0.02% polyvinylpyrrolidone/0.02% Ficoll/0.02% BSA), 5× SSC, and 0.5% SDS.

    Article Title: Close, stable homolog juxtaposition during meiosis in budding yeast is dependent on meiotic recombination, occurs independently of synapsis, and is distinct from DSB-independent pairing contacts
    Article Snippet: .. Probe g was then reacted with Cy3-NHS ester dye (Amersham, catalog no. PA23001) per the manufacturer's instructions except that ∼5 μg of DNA was added to the dye reaction and incubated at 37°C for 2 h before quenching with 37.5 mM glycine (pH 8), followed by a further incubation at 37°C for 10 min. Probe q was labeled in the presence of 40 μM Alexa 488-dUTP (Molecular Probes) by nick translation using DNA Polymerase I/DNaseI (GIBCO BRL) in a reaction containing 50 mM Tris-HCl (pH 7.8), 5 mM MgCl2 , 10 mM 2-mercaptoethanol, 10 μg/mL BSA, 40 μM dATP, dCTP, and dGTP, 8 μM dTTP with an additional 4 U/μL DNase (GIBCO) at 15°C for 1 h. Reactions were stopped by the addition of EDTA (pH 8.0) to a final concentration of 50 mM and heated to 65°C for 15 min. Each probe was purified through a G-50 Sephadex spin column prior to slide hybridization. .. Hybridization and washing of probes was carried out essentially as described in except that 0.4× SSC, 1% BSA, and 4% dextran sulfate were used for the hybridization and no detection antibodies were used.

    BAC Assay:

    Article Title: Single copies of mutant KRAS and mutant PIK3CA cooperate in immortalized human epithelial cells to induce tumor formation
    Article Snippet: .. For probes, DNA was extracted from BAC clones and labeled by nick translation with Green dUTP or Red dUTP (Abbott Laboratories) using DNA polymerase/DNase I (Invitrogen). ..

    Hybridization:

    Article Title: Close, stable homolog juxtaposition during meiosis in budding yeast is dependent on meiotic recombination, occurs independently of synapsis, and is distinct from DSB-independent pairing contacts
    Article Snippet: .. Probe g was then reacted with Cy3-NHS ester dye (Amersham, catalog no. PA23001) per the manufacturer's instructions except that ∼5 μg of DNA was added to the dye reaction and incubated at 37°C for 2 h before quenching with 37.5 mM glycine (pH 8), followed by a further incubation at 37°C for 10 min. Probe q was labeled in the presence of 40 μM Alexa 488-dUTP (Molecular Probes) by nick translation using DNA Polymerase I/DNaseI (GIBCO BRL) in a reaction containing 50 mM Tris-HCl (pH 7.8), 5 mM MgCl2 , 10 mM 2-mercaptoethanol, 10 μg/mL BSA, 40 μM dATP, dCTP, and dGTP, 8 μM dTTP with an additional 4 U/μL DNase (GIBCO) at 15°C for 1 h. Reactions were stopped by the addition of EDTA (pH 8.0) to a final concentration of 50 mM and heated to 65°C for 15 min. Each probe was purified through a G-50 Sephadex spin column prior to slide hybridization. .. Hybridization and washing of probes was carried out essentially as described in except that 0.4× SSC, 1% BSA, and 4% dextran sulfate were used for the hybridization and no detection antibodies were used.

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    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, 2020-05
    99/100 stars
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    99
    Thermo Fisher dnase i reaction buffer
    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)
    Dnase I Reaction Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i reaction buffer/product/Thermo Fisher
    Average 99 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    dnase i reaction buffer - by Bioz Stars, 2020-05
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    99
    Thermo Fisher dnase i amplification grade
    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)
    Dnase I Amplification Grade, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i amplification grade/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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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

    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: Next, 0.5 μL of genomic DNA digestion mix (0.1 U of DNase I Amplification Grade (Thermo Fisher) and 2× DNase I Reaction Buffer (Thermo Fisher) in RNase-free water) was added to 1 μL of the single-cell lysate in a 96-well PCR plate and incubated at 25 °C for 5 min. After genomic DNA digestion, we added 0.5 µL of denaturing mix (8 mM EDTA and 0.02% NP40 in RNase-free water) to the digested sample, followed by incubation at 70 °C for 5 min to inactivate DNase I and desaturate the RNAs.

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

    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: A mixture containing 2 μL of conventional RT mix (1.5× PrimeScript buffer, 0.6 pmol oligo(dT)18 (Thermo Fisher), 8 pmol random hexamers (TaKaRa), and 1.5× PrimeScript enzyme mix in RNase-free water) or 2 μL of RT-RamDA mix (1.5× PrimeScript buffer, 0.6 pmol oligo(dT)18, 8 pmol random hexamers or NSRs, 0.2 U of DNase I Amplification Grade (Thermo Fisher), 100 ng of T4 gene 32 protein (Roche), and 1.5× PrimeScript enzyme mix in RNase-free water) was added to 1 μL of diluted, denatured template RNA.

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