draiii hf  (New England Biolabs)


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    New England Biolabs draiii hf
    a In vitro transcription of linear DNA containing 12 CTG tandem repeats. <t>A</t> <t>circular</t> DNA construct contains a single T7RNAP promoter, the OriC, 12 CTG tandem repeats, and a <t>DraIII</t> restriction site. The circular DNA construct was linearized using DraIII, by cutting upstream of the T7RNAP promoter. The linear DNA was in vitro transcribed using T7RNAP. b Agarose gel electrophoresis of transcribed linear DNA indicates the presence of two RNA species (lane 4), one in which the RNA polymerase transcribes the entire linear DNA (END) and RNA of unknown nature. With nanopore sensing, it is later revealed that the lower band corresponds to RNA, where transcription is prematurely terminated within the OriC sequence (PT). Gel lanes: 1 – 1 kbp ladder; 2 – single-stranded RNA ladder; 3 – transcribed RNA from linear DNA; 4 - transcribed RNA from linear DNA treated with DNase I. Transcriptions were performed in triplicate. c RNA transcripts were hybridized with short complementary DNA oligonucleotides (~40 nt), producing RNA IDs. CUG repeats in RNA were labeled with two streptavidins (repeats label ”R”), indicating the beginning of transcription given their vicinity to the promoter. Further positioning along the transcript is achieved using bits “1” that have one streptavidin enabling their distinction from label “R”. d RNA IDs made from RNA transcripts that have different termination points (PT or END) translocating through the nanopore. Nanopore readout is based on electrophoretically driven transport of negatively charged RNA IDs through the nanopore towards a positively charged electrode. e Nanopore RNA ID readouts for PT and END RNAs are presented left and right, respectively. RNA ID for both PT and END shows downward spikes associated to the labeled repeats “R” (red) and “1” bits (blue). PT translocation finishes right after the second “1” bit hence making a clear distinction towards END translocations, which takes longer to translocate through the pore. Source data are provided as a Source Data file.
    Draiii Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination"

    Article Title: Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination

    Journal: Nature Communications

    doi: 10.1038/s41467-024-45968-8

    a In vitro transcription of linear DNA containing 12 CTG tandem repeats. A circular DNA construct contains a single T7RNAP promoter, the OriC, 12 CTG tandem repeats, and a DraIII restriction site. The circular DNA construct was linearized using DraIII, by cutting upstream of the T7RNAP promoter. The linear DNA was in vitro transcribed using T7RNAP. b Agarose gel electrophoresis of transcribed linear DNA indicates the presence of two RNA species (lane 4), one in which the RNA polymerase transcribes the entire linear DNA (END) and RNA of unknown nature. With nanopore sensing, it is later revealed that the lower band corresponds to RNA, where transcription is prematurely terminated within the OriC sequence (PT). Gel lanes: 1 – 1 kbp ladder; 2 – single-stranded RNA ladder; 3 – transcribed RNA from linear DNA; 4 - transcribed RNA from linear DNA treated with DNase I. Transcriptions were performed in triplicate. c RNA transcripts were hybridized with short complementary DNA oligonucleotides (~40 nt), producing RNA IDs. CUG repeats in RNA were labeled with two streptavidins (repeats label ”R”), indicating the beginning of transcription given their vicinity to the promoter. Further positioning along the transcript is achieved using bits “1” that have one streptavidin enabling their distinction from label “R”. d RNA IDs made from RNA transcripts that have different termination points (PT or END) translocating through the nanopore. Nanopore readout is based on electrophoretically driven transport of negatively charged RNA IDs through the nanopore towards a positively charged electrode. e Nanopore RNA ID readouts for PT and END RNAs are presented left and right, respectively. RNA ID for both PT and END shows downward spikes associated to the labeled repeats “R” (red) and “1” bits (blue). PT translocation finishes right after the second “1” bit hence making a clear distinction towards END translocations, which takes longer to translocate through the pore. Source data are provided as a Source Data file.
    Figure Legend Snippet: a In vitro transcription of linear DNA containing 12 CTG tandem repeats. A circular DNA construct contains a single T7RNAP promoter, the OriC, 12 CTG tandem repeats, and a DraIII restriction site. The circular DNA construct was linearized using DraIII, by cutting upstream of the T7RNAP promoter. The linear DNA was in vitro transcribed using T7RNAP. b Agarose gel electrophoresis of transcribed linear DNA indicates the presence of two RNA species (lane 4), one in which the RNA polymerase transcribes the entire linear DNA (END) and RNA of unknown nature. With nanopore sensing, it is later revealed that the lower band corresponds to RNA, where transcription is prematurely terminated within the OriC sequence (PT). Gel lanes: 1 – 1 kbp ladder; 2 – single-stranded RNA ladder; 3 – transcribed RNA from linear DNA; 4 - transcribed RNA from linear DNA treated with DNase I. Transcriptions were performed in triplicate. c RNA transcripts were hybridized with short complementary DNA oligonucleotides (~40 nt), producing RNA IDs. CUG repeats in RNA were labeled with two streptavidins (repeats label ”R”), indicating the beginning of transcription given their vicinity to the promoter. Further positioning along the transcript is achieved using bits “1” that have one streptavidin enabling their distinction from label “R”. d RNA IDs made from RNA transcripts that have different termination points (PT or END) translocating through the nanopore. Nanopore readout is based on electrophoretically driven transport of negatively charged RNA IDs through the nanopore towards a positively charged electrode. e Nanopore RNA ID readouts for PT and END RNAs are presented left and right, respectively. RNA ID for both PT and END shows downward spikes associated to the labeled repeats “R” (red) and “1” bits (blue). PT translocation finishes right after the second “1” bit hence making a clear distinction towards END translocations, which takes longer to translocate through the pore. Source data are provided as a Source Data file.

    Techniques Used: In Vitro, Construct, Agarose Gel Electrophoresis, Sequencing, Labeling, Translocation Assay

    draiii hf  (New England Biolabs)


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    New England Biolabs draiii hf
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    draiii hf  (New England Biolabs)


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    New England Biolabs draiii hf
    a In vitro transcription of linear DNA containing 12 CTG tandem repeats. <t>A</t> <t>circular</t> DNA construct contains a single T7RNAP promoter, the OriC, 12 CTG tandem repeats, and a <t>DraIII</t> restriction site. The circular DNA construct was linearized using DraIII, by cutting upstream of the T7RNAP promoter. The linear DNA was in vitro transcribed using T7RNAP. b Agarose gel electrophoresis of transcribed linear DNA indicates the presence of two RNA species (lane 4), one in which the RNA polymerase transcribes the entire linear DNA (END) and RNA of unknown nature. With nanopore sensing, it is later revealed that the lower band corresponds to RNA, where transcription is prematurely terminated within the OriC sequence (PT). Gel lanes: 1 – 1 kbp ladder; 2 – single-stranded RNA ladder; 3 – transcribed RNA from linear DNA; 4 - transcribed RNA from linear DNA treated with DNase I. Transcriptions were performed in triplicate. c RNA transcripts were hybridized with short complementary DNA oligonucleotides (~40 nt), producing RNA IDs. CUG repeats in RNA were labeled with two streptavidins (repeats label ”R”), indicating the beginning of transcription given their vicinity to the promoter. Further positioning along the transcript is achieved using bits “1” that have one streptavidin enabling their distinction from label “R”. d RNA IDs made from RNA transcripts that have different termination points (PT or END) translocating through the nanopore. Nanopore readout is based on electrophoretically driven transport of negatively charged RNA IDs through the nanopore towards a positively charged electrode. e Nanopore RNA ID readouts for PT and END RNAs are presented left and right, respectively. RNA ID for both PT and END shows downward spikes associated to the labeled repeats “R” (red) and “1” bits (blue). PT translocation finishes right after the second “1” bit hence making a clear distinction towards END translocations, which takes longer to translocate through the pore. Source data are provided as a Source Data file.
    Draiii Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination"

    Article Title: Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination

    Journal: Nature Communications

    doi: 10.1038/s41467-024-45968-8

    a In vitro transcription of linear DNA containing 12 CTG tandem repeats. A circular DNA construct contains a single T7RNAP promoter, the OriC, 12 CTG tandem repeats, and a DraIII restriction site. The circular DNA construct was linearized using DraIII, by cutting upstream of the T7RNAP promoter. The linear DNA was in vitro transcribed using T7RNAP. b Agarose gel electrophoresis of transcribed linear DNA indicates the presence of two RNA species (lane 4), one in which the RNA polymerase transcribes the entire linear DNA (END) and RNA of unknown nature. With nanopore sensing, it is later revealed that the lower band corresponds to RNA, where transcription is prematurely terminated within the OriC sequence (PT). Gel lanes: 1 – 1 kbp ladder; 2 – single-stranded RNA ladder; 3 – transcribed RNA from linear DNA; 4 - transcribed RNA from linear DNA treated with DNase I. Transcriptions were performed in triplicate. c RNA transcripts were hybridized with short complementary DNA oligonucleotides (~40 nt), producing RNA IDs. CUG repeats in RNA were labeled with two streptavidins (repeats label ”R”), indicating the beginning of transcription given their vicinity to the promoter. Further positioning along the transcript is achieved using bits “1” that have one streptavidin enabling their distinction from label “R”. d RNA IDs made from RNA transcripts that have different termination points (PT or END) translocating through the nanopore. Nanopore readout is based on electrophoretically driven transport of negatively charged RNA IDs through the nanopore towards a positively charged electrode. e Nanopore RNA ID readouts for PT and END RNAs are presented left and right, respectively. RNA ID for both PT and END shows downward spikes associated to the labeled repeats “R” (red) and “1” bits (blue). PT translocation finishes right after the second “1” bit hence making a clear distinction towards END translocations, which takes longer to translocate through the pore. Source data are provided as a Source Data file.
    Figure Legend Snippet: a In vitro transcription of linear DNA containing 12 CTG tandem repeats. A circular DNA construct contains a single T7RNAP promoter, the OriC, 12 CTG tandem repeats, and a DraIII restriction site. The circular DNA construct was linearized using DraIII, by cutting upstream of the T7RNAP promoter. The linear DNA was in vitro transcribed using T7RNAP. b Agarose gel electrophoresis of transcribed linear DNA indicates the presence of two RNA species (lane 4), one in which the RNA polymerase transcribes the entire linear DNA (END) and RNA of unknown nature. With nanopore sensing, it is later revealed that the lower band corresponds to RNA, where transcription is prematurely terminated within the OriC sequence (PT). Gel lanes: 1 – 1 kbp ladder; 2 – single-stranded RNA ladder; 3 – transcribed RNA from linear DNA; 4 - transcribed RNA from linear DNA treated with DNase I. Transcriptions were performed in triplicate. c RNA transcripts were hybridized with short complementary DNA oligonucleotides (~40 nt), producing RNA IDs. CUG repeats in RNA were labeled with two streptavidins (repeats label ”R”), indicating the beginning of transcription given their vicinity to the promoter. Further positioning along the transcript is achieved using bits “1” that have one streptavidin enabling their distinction from label “R”. d RNA IDs made from RNA transcripts that have different termination points (PT or END) translocating through the nanopore. Nanopore readout is based on electrophoretically driven transport of negatively charged RNA IDs through the nanopore towards a positively charged electrode. e Nanopore RNA ID readouts for PT and END RNAs are presented left and right, respectively. RNA ID for both PT and END shows downward spikes associated to the labeled repeats “R” (red) and “1” bits (blue). PT translocation finishes right after the second “1” bit hence making a clear distinction towards END translocations, which takes longer to translocate through the pore. Source data are provided as a Source Data file.

    Techniques Used: In Vitro, Construct, Agarose Gel Electrophoresis, Sequencing, Labeling, Translocation Assay

    draiii hf treatment  (New England Biolabs)


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    New England Biolabs draiii hf treatment
    a In vitro transcription of <t>linear</t> <t>DNA</t> containing 12 CTG tandem repeats. A circular DNA construct contains a single T7RNAP promoter, the OriC, 12 CTG tandem repeats, and a <t>DraIII</t> restriction site. The circular DNA construct was linearized using DraIII, by cutting upstream of the T7RNAP promoter. The linear DNA was in vitro transcribed using T7RNAP. b Agarose gel electrophoresis of transcribed linear DNA indicates the presence of two RNA species (lane 4), one in which the RNA polymerase transcribes the entire linear DNA (END) and RNA of unknown nature. With nanopore sensing, it is later revealed that the lower band corresponds to RNA, where transcription is prematurely terminated within the OriC sequence (PT). Gel lanes: 1 – 1 kbp ladder; 2 – single-stranded RNA ladder; 3 – transcribed RNA from linear DNA; 4 - transcribed RNA from linear DNA treated with DNase I. Transcriptions were performed in triplicate. c RNA transcripts were hybridized with short complementary DNA oligonucleotides (~40 nt), producing RNA IDs. CUG repeats in RNA were labeled with two streptavidins (repeats label ”R”), indicating the beginning of transcription given their vicinity to the promoter. Further positioning along the transcript is achieved using bits “1” that have one streptavidin enabling their distinction from label “R”. d RNA IDs made from RNA transcripts that have different termination points (PT or END) translocating through the nanopore. Nanopore readout is based on electrophoretically driven transport of negatively charged RNA IDs through the nanopore towards a positively charged electrode. e Nanopore RNA ID readouts for PT and END RNAs are presented left and right, respectively. RNA ID for both PT and END shows downward spikes associated to the labeled repeats “R” (red) and “1” bits (blue). PT translocation finishes right after the second “1” bit hence making a clear distinction towards END translocations, which takes longer to translocate through the pore. Source data are provided as a Source Data file.
    Draiii Hf Treatment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination"

    Article Title: Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination

    Journal: Nature Communications

    doi: 10.1038/s41467-024-45968-8

    a In vitro transcription of linear DNA containing 12 CTG tandem repeats. A circular DNA construct contains a single T7RNAP promoter, the OriC, 12 CTG tandem repeats, and a DraIII restriction site. The circular DNA construct was linearized using DraIII, by cutting upstream of the T7RNAP promoter. The linear DNA was in vitro transcribed using T7RNAP. b Agarose gel electrophoresis of transcribed linear DNA indicates the presence of two RNA species (lane 4), one in which the RNA polymerase transcribes the entire linear DNA (END) and RNA of unknown nature. With nanopore sensing, it is later revealed that the lower band corresponds to RNA, where transcription is prematurely terminated within the OriC sequence (PT). Gel lanes: 1 – 1 kbp ladder; 2 – single-stranded RNA ladder; 3 – transcribed RNA from linear DNA; 4 - transcribed RNA from linear DNA treated with DNase I. Transcriptions were performed in triplicate. c RNA transcripts were hybridized with short complementary DNA oligonucleotides (~40 nt), producing RNA IDs. CUG repeats in RNA were labeled with two streptavidins (repeats label ”R”), indicating the beginning of transcription given their vicinity to the promoter. Further positioning along the transcript is achieved using bits “1” that have one streptavidin enabling their distinction from label “R”. d RNA IDs made from RNA transcripts that have different termination points (PT or END) translocating through the nanopore. Nanopore readout is based on electrophoretically driven transport of negatively charged RNA IDs through the nanopore towards a positively charged electrode. e Nanopore RNA ID readouts for PT and END RNAs are presented left and right, respectively. RNA ID for both PT and END shows downward spikes associated to the labeled repeats “R” (red) and “1” bits (blue). PT translocation finishes right after the second “1” bit hence making a clear distinction towards END translocations, which takes longer to translocate through the pore. Source data are provided as a Source Data file.
    Figure Legend Snippet: a In vitro transcription of linear DNA containing 12 CTG tandem repeats. A circular DNA construct contains a single T7RNAP promoter, the OriC, 12 CTG tandem repeats, and a DraIII restriction site. The circular DNA construct was linearized using DraIII, by cutting upstream of the T7RNAP promoter. The linear DNA was in vitro transcribed using T7RNAP. b Agarose gel electrophoresis of transcribed linear DNA indicates the presence of two RNA species (lane 4), one in which the RNA polymerase transcribes the entire linear DNA (END) and RNA of unknown nature. With nanopore sensing, it is later revealed that the lower band corresponds to RNA, where transcription is prematurely terminated within the OriC sequence (PT). Gel lanes: 1 – 1 kbp ladder; 2 – single-stranded RNA ladder; 3 – transcribed RNA from linear DNA; 4 - transcribed RNA from linear DNA treated with DNase I. Transcriptions were performed in triplicate. c RNA transcripts were hybridized with short complementary DNA oligonucleotides (~40 nt), producing RNA IDs. CUG repeats in RNA were labeled with two streptavidins (repeats label ”R”), indicating the beginning of transcription given their vicinity to the promoter. Further positioning along the transcript is achieved using bits “1” that have one streptavidin enabling their distinction from label “R”. d RNA IDs made from RNA transcripts that have different termination points (PT or END) translocating through the nanopore. Nanopore readout is based on electrophoretically driven transport of negatively charged RNA IDs through the nanopore towards a positively charged electrode. e Nanopore RNA ID readouts for PT and END RNAs are presented left and right, respectively. RNA ID for both PT and END shows downward spikes associated to the labeled repeats “R” (red) and “1” bits (blue). PT translocation finishes right after the second “1” bit hence making a clear distinction towards END translocations, which takes longer to translocate through the pore. Source data are provided as a Source Data file.

    Techniques Used: In Vitro, Construct, Agarose Gel Electrophoresis, Sequencing, Labeling, Translocation Assay

    r3510s  (New England Biolabs)


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    draiii hf  (New England Biolabs)


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    New England Biolabs draiii hf
    a In vitro transcription of linear DNA containing 12 CTG tandem repeats. <t>A</t> <t>circular</t> DNA construct contains a single T7RNAP promoter, the OriC, 12 CTG tandem repeats, and a <t>DraIII</t> restriction site. The circular DNA construct was linearized using DraIII, by cutting upstream of the T7RNAP promoter. The linear DNA was in vitro transcribed using T7RNAP. b Agarose gel electrophoresis of transcribed linear DNA indicates the presence of two RNA species (lane 4), one in which the RNA polymerase transcribes the entire linear DNA (END) and RNA of unknown nature. With nanopore sensing, it is later revealed that the lower band corresponds to RNA, where transcription is prematurely terminated within the OriC sequence (PT). Gel lanes: 1 – 1 kbp ladder; 2 – single-stranded RNA ladder; 3 – transcribed RNA from linear DNA; 4 - transcribed RNA from linear DNA treated with DNase I. Transcriptions were performed in triplicate. c RNA transcripts were hybridized with short complementary DNA oligonucleotides (~40 nt), producing RNA IDs. CUG repeats in RNA were labeled with two streptavidins (repeats label ”R”), indicating the beginning of transcription given their vicinity to the promoter. Further positioning along the transcript is achieved using bits “1” that have one streptavidin enabling their distinction from label “R”. d RNA IDs made from RNA transcripts that have different termination points (PT or END) translocating through the nanopore. Nanopore readout is based on electrophoretically driven transport of negatively charged RNA IDs through the nanopore towards a positively charged electrode. e Nanopore RNA ID readouts for PT and END RNAs are presented left and right, respectively. RNA ID for both PT and END shows downward spikes associated to the labeled repeats “R” (red) and “1” bits (blue). PT translocation finishes right after the second “1” bit hence making a clear distinction towards END translocations, which takes longer to translocate through the pore. Source data are provided as a Source Data file.
    Draiii Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs draiii hf treatment
    a In vitro transcription of <t>linear</t> <t>DNA</t> containing 12 CTG tandem repeats. A circular DNA construct contains a single T7RNAP promoter, the OriC, 12 CTG tandem repeats, and a <t>DraIII</t> restriction site. The circular DNA construct was linearized using DraIII, by cutting upstream of the T7RNAP promoter. The linear DNA was in vitro transcribed using T7RNAP. b Agarose gel electrophoresis of transcribed linear DNA indicates the presence of two RNA species (lane 4), one in which the RNA polymerase transcribes the entire linear DNA (END) and RNA of unknown nature. With nanopore sensing, it is later revealed that the lower band corresponds to RNA, where transcription is prematurely terminated within the OriC sequence (PT). Gel lanes: 1 – 1 kbp ladder; 2 – single-stranded RNA ladder; 3 – transcribed RNA from linear DNA; 4 - transcribed RNA from linear DNA treated with DNase I. Transcriptions were performed in triplicate. c RNA transcripts were hybridized with short complementary DNA oligonucleotides (~40 nt), producing RNA IDs. CUG repeats in RNA were labeled with two streptavidins (repeats label ”R”), indicating the beginning of transcription given their vicinity to the promoter. Further positioning along the transcript is achieved using bits “1” that have one streptavidin enabling their distinction from label “R”. d RNA IDs made from RNA transcripts that have different termination points (PT or END) translocating through the nanopore. Nanopore readout is based on electrophoretically driven transport of negatively charged RNA IDs through the nanopore towards a positively charged electrode. e Nanopore RNA ID readouts for PT and END RNAs are presented left and right, respectively. RNA ID for both PT and END shows downward spikes associated to the labeled repeats “R” (red) and “1” bits (blue). PT translocation finishes right after the second “1” bit hence making a clear distinction towards END translocations, which takes longer to translocate through the pore. Source data are provided as a Source Data file.
    Draiii Hf Treatment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs r3510s
    a In vitro transcription of <t>linear</t> <t>DNA</t> containing 12 CTG tandem repeats. A circular DNA construct contains a single T7RNAP promoter, the OriC, 12 CTG tandem repeats, and a <t>DraIII</t> restriction site. The circular DNA construct was linearized using DraIII, by cutting upstream of the T7RNAP promoter. The linear DNA was in vitro transcribed using T7RNAP. b Agarose gel electrophoresis of transcribed linear DNA indicates the presence of two RNA species (lane 4), one in which the RNA polymerase transcribes the entire linear DNA (END) and RNA of unknown nature. With nanopore sensing, it is later revealed that the lower band corresponds to RNA, where transcription is prematurely terminated within the OriC sequence (PT). Gel lanes: 1 – 1 kbp ladder; 2 – single-stranded RNA ladder; 3 – transcribed RNA from linear DNA; 4 - transcribed RNA from linear DNA treated with DNase I. Transcriptions were performed in triplicate. c RNA transcripts were hybridized with short complementary DNA oligonucleotides (~40 nt), producing RNA IDs. CUG repeats in RNA were labeled with two streptavidins (repeats label ”R”), indicating the beginning of transcription given their vicinity to the promoter. Further positioning along the transcript is achieved using bits “1” that have one streptavidin enabling their distinction from label “R”. d RNA IDs made from RNA transcripts that have different termination points (PT or END) translocating through the nanopore. Nanopore readout is based on electrophoretically driven transport of negatively charged RNA IDs through the nanopore towards a positively charged electrode. e Nanopore RNA ID readouts for PT and END RNAs are presented left and right, respectively. RNA ID for both PT and END shows downward spikes associated to the labeled repeats “R” (red) and “1” bits (blue). PT translocation finishes right after the second “1” bit hence making a clear distinction towards END translocations, which takes longer to translocate through the pore. Source data are provided as a Source Data file.
    R3510s, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs draiii
    a In vitro transcription of <t>linear</t> <t>DNA</t> containing 12 CTG tandem repeats. A circular DNA construct contains a single T7RNAP promoter, the OriC, 12 CTG tandem repeats, and a <t>DraIII</t> restriction site. The circular DNA construct was linearized using DraIII, by cutting upstream of the T7RNAP promoter. The linear DNA was in vitro transcribed using T7RNAP. b Agarose gel electrophoresis of transcribed linear DNA indicates the presence of two RNA species (lane 4), one in which the RNA polymerase transcribes the entire linear DNA (END) and RNA of unknown nature. With nanopore sensing, it is later revealed that the lower band corresponds to RNA, where transcription is prematurely terminated within the OriC sequence (PT). Gel lanes: 1 – 1 kbp ladder; 2 – single-stranded RNA ladder; 3 – transcribed RNA from linear DNA; 4 - transcribed RNA from linear DNA treated with DNase I. Transcriptions were performed in triplicate. c RNA transcripts were hybridized with short complementary DNA oligonucleotides (~40 nt), producing RNA IDs. CUG repeats in RNA were labeled with two streptavidins (repeats label ”R”), indicating the beginning of transcription given their vicinity to the promoter. Further positioning along the transcript is achieved using bits “1” that have one streptavidin enabling their distinction from label “R”. d RNA IDs made from RNA transcripts that have different termination points (PT or END) translocating through the nanopore. Nanopore readout is based on electrophoretically driven transport of negatively charged RNA IDs through the nanopore towards a positively charged electrode. e Nanopore RNA ID readouts for PT and END RNAs are presented left and right, respectively. RNA ID for both PT and END shows downward spikes associated to the labeled repeats “R” (red) and “1” bits (blue). PT translocation finishes right after the second “1” bit hence making a clear distinction towards END translocations, which takes longer to translocate through the pore. Source data are provided as a Source Data file.
    Draiii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs draiii-hf
    a In vitro transcription of <t>linear</t> <t>DNA</t> containing 12 CTG tandem repeats. A circular DNA construct contains a single T7RNAP promoter, the OriC, 12 CTG tandem repeats, and a <t>DraIII</t> restriction site. The circular DNA construct was linearized using DraIII, by cutting upstream of the T7RNAP promoter. The linear DNA was in vitro transcribed using T7RNAP. b Agarose gel electrophoresis of transcribed linear DNA indicates the presence of two RNA species (lane 4), one in which the RNA polymerase transcribes the entire linear DNA (END) and RNA of unknown nature. With nanopore sensing, it is later revealed that the lower band corresponds to RNA, where transcription is prematurely terminated within the OriC sequence (PT). Gel lanes: 1 – 1 kbp ladder; 2 – single-stranded RNA ladder; 3 – transcribed RNA from linear DNA; 4 - transcribed RNA from linear DNA treated with DNase I. Transcriptions were performed in triplicate. c RNA transcripts were hybridized with short complementary DNA oligonucleotides (~40 nt), producing RNA IDs. CUG repeats in RNA were labeled with two streptavidins (repeats label ”R”), indicating the beginning of transcription given their vicinity to the promoter. Further positioning along the transcript is achieved using bits “1” that have one streptavidin enabling their distinction from label “R”. d RNA IDs made from RNA transcripts that have different termination points (PT or END) translocating through the nanopore. Nanopore readout is based on electrophoretically driven transport of negatively charged RNA IDs through the nanopore towards a positively charged electrode. e Nanopore RNA ID readouts for PT and END RNAs are presented left and right, respectively. RNA ID for both PT and END shows downward spikes associated to the labeled repeats “R” (red) and “1” bits (blue). PT translocation finishes right after the second “1” bit hence making a clear distinction towards END translocations, which takes longer to translocate through the pore. Source data are provided as a Source Data file.
    Draiii Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs draiii hf restriction enzyme
    a In vitro transcription of <t>linear</t> <t>DNA</t> containing 12 CTG tandem repeats. A circular DNA construct contains a single T7RNAP promoter, the OriC, 12 CTG tandem repeats, and a <t>DraIII</t> restriction site. The circular DNA construct was linearized using DraIII, by cutting upstream of the T7RNAP promoter. The linear DNA was in vitro transcribed using T7RNAP. b Agarose gel electrophoresis of transcribed linear DNA indicates the presence of two RNA species (lane 4), one in which the RNA polymerase transcribes the entire linear DNA (END) and RNA of unknown nature. With nanopore sensing, it is later revealed that the lower band corresponds to RNA, where transcription is prematurely terminated within the OriC sequence (PT). Gel lanes: 1 – 1 kbp ladder; 2 – single-stranded RNA ladder; 3 – transcribed RNA from linear DNA; 4 - transcribed RNA from linear DNA treated with DNase I. Transcriptions were performed in triplicate. c RNA transcripts were hybridized with short complementary DNA oligonucleotides (~40 nt), producing RNA IDs. CUG repeats in RNA were labeled with two streptavidins (repeats label ”R”), indicating the beginning of transcription given their vicinity to the promoter. Further positioning along the transcript is achieved using bits “1” that have one streptavidin enabling their distinction from label “R”. d RNA IDs made from RNA transcripts that have different termination points (PT or END) translocating through the nanopore. Nanopore readout is based on electrophoretically driven transport of negatively charged RNA IDs through the nanopore towards a positively charged electrode. e Nanopore RNA ID readouts for PT and END RNAs are presented left and right, respectively. RNA ID for both PT and END shows downward spikes associated to the labeled repeats “R” (red) and “1” bits (blue). PT translocation finishes right after the second “1” bit hence making a clear distinction towards END translocations, which takes longer to translocate through the pore. Source data are provided as a Source Data file.
    Draiii Hf Restriction Enzyme, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    a In vitro transcription of linear DNA containing 12 CTG tandem repeats. A circular DNA construct contains a single T7RNAP promoter, the OriC, 12 CTG tandem repeats, and a DraIII restriction site. The circular DNA construct was linearized using DraIII, by cutting upstream of the T7RNAP promoter. The linear DNA was in vitro transcribed using T7RNAP. b Agarose gel electrophoresis of transcribed linear DNA indicates the presence of two RNA species (lane 4), one in which the RNA polymerase transcribes the entire linear DNA (END) and RNA of unknown nature. With nanopore sensing, it is later revealed that the lower band corresponds to RNA, where transcription is prematurely terminated within the OriC sequence (PT). Gel lanes: 1 – 1 kbp ladder; 2 – single-stranded RNA ladder; 3 – transcribed RNA from linear DNA; 4 - transcribed RNA from linear DNA treated with DNase I. Transcriptions were performed in triplicate. c RNA transcripts were hybridized with short complementary DNA oligonucleotides (~40 nt), producing RNA IDs. CUG repeats in RNA were labeled with two streptavidins (repeats label ”R”), indicating the beginning of transcription given their vicinity to the promoter. Further positioning along the transcript is achieved using bits “1” that have one streptavidin enabling their distinction from label “R”. d RNA IDs made from RNA transcripts that have different termination points (PT or END) translocating through the nanopore. Nanopore readout is based on electrophoretically driven transport of negatively charged RNA IDs through the nanopore towards a positively charged electrode. e Nanopore RNA ID readouts for PT and END RNAs are presented left and right, respectively. RNA ID for both PT and END shows downward spikes associated to the labeled repeats “R” (red) and “1” bits (blue). PT translocation finishes right after the second “1” bit hence making a clear distinction towards END translocations, which takes longer to translocate through the pore. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination

    doi: 10.1038/s41467-024-45968-8

    Figure Lengend Snippet: a In vitro transcription of linear DNA containing 12 CTG tandem repeats. A circular DNA construct contains a single T7RNAP promoter, the OriC, 12 CTG tandem repeats, and a DraIII restriction site. The circular DNA construct was linearized using DraIII, by cutting upstream of the T7RNAP promoter. The linear DNA was in vitro transcribed using T7RNAP. b Agarose gel electrophoresis of transcribed linear DNA indicates the presence of two RNA species (lane 4), one in which the RNA polymerase transcribes the entire linear DNA (END) and RNA of unknown nature. With nanopore sensing, it is later revealed that the lower band corresponds to RNA, where transcription is prematurely terminated within the OriC sequence (PT). Gel lanes: 1 – 1 kbp ladder; 2 – single-stranded RNA ladder; 3 – transcribed RNA from linear DNA; 4 - transcribed RNA from linear DNA treated with DNase I. Transcriptions were performed in triplicate. c RNA transcripts were hybridized with short complementary DNA oligonucleotides (~40 nt), producing RNA IDs. CUG repeats in RNA were labeled with two streptavidins (repeats label ”R”), indicating the beginning of transcription given their vicinity to the promoter. Further positioning along the transcript is achieved using bits “1” that have one streptavidin enabling their distinction from label “R”. d RNA IDs made from RNA transcripts that have different termination points (PT or END) translocating through the nanopore. Nanopore readout is based on electrophoretically driven transport of negatively charged RNA IDs through the nanopore towards a positively charged electrode. e Nanopore RNA ID readouts for PT and END RNAs are presented left and right, respectively. RNA ID for both PT and END shows downward spikes associated to the labeled repeats “R” (red) and “1” bits (blue). PT translocation finishes right after the second “1” bit hence making a clear distinction towards END translocations, which takes longer to translocate through the pore. Source data are provided as a Source Data file.

    Article Snippet: DraIII-HF (New England Biolabs (NEB), R3510S) was used to linearize the circular DNA plasmid (sequence in Supplementary Table ) following the manufacturer’s recommendations.

    Techniques: In Vitro, Construct, Agarose Gel Electrophoresis, Sequencing, Labeling, Translocation Assay

    a In vitro transcription of linear DNA containing 12 CTG tandem repeats. A circular DNA construct contains a single T7RNAP promoter, the OriC, 12 CTG tandem repeats, and a DraIII restriction site. The circular DNA construct was linearized using DraIII, by cutting upstream of the T7RNAP promoter. The linear DNA was in vitro transcribed using T7RNAP. b Agarose gel electrophoresis of transcribed linear DNA indicates the presence of two RNA species (lane 4), one in which the RNA polymerase transcribes the entire linear DNA (END) and RNA of unknown nature. With nanopore sensing, it is later revealed that the lower band corresponds to RNA, where transcription is prematurely terminated within the OriC sequence (PT). Gel lanes: 1 – 1 kbp ladder; 2 – single-stranded RNA ladder; 3 – transcribed RNA from linear DNA; 4 - transcribed RNA from linear DNA treated with DNase I. Transcriptions were performed in triplicate. c RNA transcripts were hybridized with short complementary DNA oligonucleotides (~40 nt), producing RNA IDs. CUG repeats in RNA were labeled with two streptavidins (repeats label ”R”), indicating the beginning of transcription given their vicinity to the promoter. Further positioning along the transcript is achieved using bits “1” that have one streptavidin enabling their distinction from label “R”. d RNA IDs made from RNA transcripts that have different termination points (PT or END) translocating through the nanopore. Nanopore readout is based on electrophoretically driven transport of negatively charged RNA IDs through the nanopore towards a positively charged electrode. e Nanopore RNA ID readouts for PT and END RNAs are presented left and right, respectively. RNA ID for both PT and END shows downward spikes associated to the labeled repeats “R” (red) and “1” bits (blue). PT translocation finishes right after the second “1” bit hence making a clear distinction towards END translocations, which takes longer to translocate through the pore. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination

    doi: 10.1038/s41467-024-45968-8

    Figure Lengend Snippet: a In vitro transcription of linear DNA containing 12 CTG tandem repeats. A circular DNA construct contains a single T7RNAP promoter, the OriC, 12 CTG tandem repeats, and a DraIII restriction site. The circular DNA construct was linearized using DraIII, by cutting upstream of the T7RNAP promoter. The linear DNA was in vitro transcribed using T7RNAP. b Agarose gel electrophoresis of transcribed linear DNA indicates the presence of two RNA species (lane 4), one in which the RNA polymerase transcribes the entire linear DNA (END) and RNA of unknown nature. With nanopore sensing, it is later revealed that the lower band corresponds to RNA, where transcription is prematurely terminated within the OriC sequence (PT). Gel lanes: 1 – 1 kbp ladder; 2 – single-stranded RNA ladder; 3 – transcribed RNA from linear DNA; 4 - transcribed RNA from linear DNA treated with DNase I. Transcriptions were performed in triplicate. c RNA transcripts were hybridized with short complementary DNA oligonucleotides (~40 nt), producing RNA IDs. CUG repeats in RNA were labeled with two streptavidins (repeats label ”R”), indicating the beginning of transcription given their vicinity to the promoter. Further positioning along the transcript is achieved using bits “1” that have one streptavidin enabling their distinction from label “R”. d RNA IDs made from RNA transcripts that have different termination points (PT or END) translocating through the nanopore. Nanopore readout is based on electrophoretically driven transport of negatively charged RNA IDs through the nanopore towards a positively charged electrode. e Nanopore RNA ID readouts for PT and END RNAs are presented left and right, respectively. RNA ID for both PT and END shows downward spikes associated to the labeled repeats “R” (red) and “1” bits (blue). PT translocation finishes right after the second “1” bit hence making a clear distinction towards END translocations, which takes longer to translocate through the pore. Source data are provided as a Source Data file.

    Article Snippet: After DraIII-HF treatment, DNA was purified using Monarch PCR & DNA Cleanup Kit (5 μg) (NEB, T1030S).

    Techniques: In Vitro, Construct, Agarose Gel Electrophoresis, Sequencing, Labeling, Translocation Assay