gel purified rna  (New England Biolabs)


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  • 91
    Name:
    RtcB Ligase
    Description:
    RtcB Ligase 25 rxns
    Catalog Number:
    m0458s
    Price:
    72
    Size:
    25 rxns
    Category:
    RNA Ligases
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    Structured Review

    New England Biolabs gel purified rna
    RtcB Ligase
    RtcB Ligase 25 rxns
    https://www.bioz.com/result/gel purified rna/product/New England Biolabs
    Average 91 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    gel purified rna - by Bioz Stars, 2020-08
    91/100 stars

    Images

    1) Product Images from "Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts"

    Article Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts

    Journal: Nature biotechnology

    doi: 10.1038/s41587-019-0090-6

    Tornado expression system generates circular RNA a , Ribozymes efficiently self-cleave during transcription reactions.. The construct containing Twister P1 and Twister P3 U2A ribozymes was transcribed in vitro and quenched with urea before running on denaturing PAGE and visualizing RNA. Fully cleaved products and the side products of cleavage accumulate efficiently and rapidly after transcription. b , Fully-cleaved products of transcription in a contain appropriate ends for circularization by the endogenous ligase, RtcB. We excised the fully-cleaved RNA from a and performed an RtcB ligation reactions. RtcB treatment produces a shift in gel mobility that is not observed without ligation or with pre-treatment with T4 PNK. This shift in gel mobility suggests that the fully-cleaved RNA contains the appropriate ends for ligation. Staining of the gel with DFHBI-1T and comparison of fluorescence relative to SYBR Gold signal demonstrates that circular Broccoli is brighter than linear Broccoli. c , Twister-based ribozyme-assisted circular RNA (racRNA) expression generates significantly higher levels of circular RNA than the previous circular RNA expressing system. HEK293T cells expressed racRNA Broccoli from a variety of racRNA expression systems (see Fig. 1 ) with different combinations of 5’ and 3’ ribozymes and were compared to expression using the tricY system. Cells were treated with actinomycin D (ActD) for 6 h to observe the drop in RNA levels after new RNA synthesis was inhibited. The Twister P1 and Twister P3 U2A construct, dubbed “Tornado”, expresses high levels of Broccoli RNA that exhibit high stability, characteristic of circRNA. d , Tornado-expressed RNA is decisively circular. DNA-directed cleavage by RNase H of a linear RNA produces two bands, each of expected size given the transcript length and probe site. The identical treatment of the same sequence expressed from Tornado produces a single band similar in size to the uncleaved transcribed sample.
    Figure Legend Snippet: Tornado expression system generates circular RNA a , Ribozymes efficiently self-cleave during transcription reactions.. The construct containing Twister P1 and Twister P3 U2A ribozymes was transcribed in vitro and quenched with urea before running on denaturing PAGE and visualizing RNA. Fully cleaved products and the side products of cleavage accumulate efficiently and rapidly after transcription. b , Fully-cleaved products of transcription in a contain appropriate ends for circularization by the endogenous ligase, RtcB. We excised the fully-cleaved RNA from a and performed an RtcB ligation reactions. RtcB treatment produces a shift in gel mobility that is not observed without ligation or with pre-treatment with T4 PNK. This shift in gel mobility suggests that the fully-cleaved RNA contains the appropriate ends for ligation. Staining of the gel with DFHBI-1T and comparison of fluorescence relative to SYBR Gold signal demonstrates that circular Broccoli is brighter than linear Broccoli. c , Twister-based ribozyme-assisted circular RNA (racRNA) expression generates significantly higher levels of circular RNA than the previous circular RNA expressing system. HEK293T cells expressed racRNA Broccoli from a variety of racRNA expression systems (see Fig. 1 ) with different combinations of 5’ and 3’ ribozymes and were compared to expression using the tricY system. Cells were treated with actinomycin D (ActD) for 6 h to observe the drop in RNA levels after new RNA synthesis was inhibited. The Twister P1 and Twister P3 U2A construct, dubbed “Tornado”, expresses high levels of Broccoli RNA that exhibit high stability, characteristic of circRNA. d , Tornado-expressed RNA is decisively circular. DNA-directed cleavage by RNase H of a linear RNA produces two bands, each of expected size given the transcript length and probe site. The identical treatment of the same sequence expressed from Tornado produces a single band similar in size to the uncleaved transcribed sample.

    Techniques Used: Expressing, Construct, In Vitro, Polyacrylamide Gel Electrophoresis, Ligation, Staining, Fluorescence, Sequencing

    2) Product Images from "Small circRNAs with self-cleaving ribozymes are frequently expressed in metazoan transcriptomes"

    Article Title: Small circRNAs with self-cleaving ribozymes are frequently expressed in metazoan transcriptomes

    Journal: bioRxiv

    doi: 10.1101/721605

    Circularization of Mexican axolotl self-cleaved retrozyme RNA. Purified linear monomeric RNA resulting from double self-cleavage of DRtz353 (see Figure 4C ) was run directly in a denaturing gel (lane 1), after 1h incubation with RtcB ligase in its corresponding buffer (lane 2), in RtcB buffer without protein ligase (lane 3), and in 50 mM Mg 2+ buffer (lane 4). Circular RNAs can be readily detected in lane 2 (up to 80% circularization for the 350 nt monomer) and lane 4 (3% self-circularization for the 350 nt monomer). Minor fractions of linear and circular dimeric RNAs are also indicated (lane 2).
    Figure Legend Snippet: Circularization of Mexican axolotl self-cleaved retrozyme RNA. Purified linear monomeric RNA resulting from double self-cleavage of DRtz353 (see Figure 4C ) was run directly in a denaturing gel (lane 1), after 1h incubation with RtcB ligase in its corresponding buffer (lane 2), in RtcB buffer without protein ligase (lane 3), and in 50 mM Mg 2+ buffer (lane 4). Circular RNAs can be readily detected in lane 2 (up to 80% circularization for the 350 nt monomer) and lane 4 (3% self-circularization for the 350 nt monomer). Minor fractions of linear and circular dimeric RNAs are also indicated (lane 2).

    Techniques Used: Purification, Incubation

    3) Product Images from "Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts"

    Article Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts

    Journal: Nature biotechnology

    doi: 10.1038/s41587-019-0090-6

    Tornado expression system generates circular RNA a , Ribozymes efficiently self-cleave during transcription reactions.. The construct containing Twister P1 and Twister P3 U2A ribozymes was transcribed in vitro and quenched with urea before running on denaturing PAGE and visualizing RNA. Fully cleaved products and the side products of cleavage accumulate efficiently and rapidly after transcription. b , Fully-cleaved products of transcription in a contain appropriate ends for circularization by the endogenous ligase, RtcB. We excised the fully-cleaved RNA from a and performed an RtcB ligation reactions. RtcB treatment produces a shift in gel mobility that is not observed without ligation or with pre-treatment with T4 PNK. This shift in gel mobility suggests that the fully-cleaved RNA contains the appropriate ends for ligation. Staining of the gel with DFHBI-1T and comparison of fluorescence relative to SYBR Gold signal demonstrates that circular Broccoli is brighter than linear Broccoli. c , Twister-based ribozyme-assisted circular RNA (racRNA) expression generates significantly higher levels of circular RNA than the previous circular RNA expressing system. HEK293T cells expressed racRNA Broccoli from a variety of racRNA expression systems (see Fig. 1 ) with different combinations of 5’ and 3’ ribozymes and were compared to expression using the tricY system. Cells were treated with actinomycin D (ActD) for 6 h to observe the drop in RNA levels after new RNA synthesis was inhibited. The Twister P1 and Twister P3 U2A construct, dubbed “Tornado”, expresses high levels of Broccoli RNA that exhibit high stability, characteristic of circRNA. d , Tornado-expressed RNA is decisively circular. DNA-directed cleavage by RNase H of a linear RNA produces two bands, each of expected size given the transcript length and probe site. The identical treatment of the same sequence expressed from Tornado produces a single band similar in size to the uncleaved transcribed sample.
    Figure Legend Snippet: Tornado expression system generates circular RNA a , Ribozymes efficiently self-cleave during transcription reactions.. The construct containing Twister P1 and Twister P3 U2A ribozymes was transcribed in vitro and quenched with urea before running on denaturing PAGE and visualizing RNA. Fully cleaved products and the side products of cleavage accumulate efficiently and rapidly after transcription. b , Fully-cleaved products of transcription in a contain appropriate ends for circularization by the endogenous ligase, RtcB. We excised the fully-cleaved RNA from a and performed an RtcB ligation reactions. RtcB treatment produces a shift in gel mobility that is not observed without ligation or with pre-treatment with T4 PNK. This shift in gel mobility suggests that the fully-cleaved RNA contains the appropriate ends for ligation. Staining of the gel with DFHBI-1T and comparison of fluorescence relative to SYBR Gold signal demonstrates that circular Broccoli is brighter than linear Broccoli. c , Twister-based ribozyme-assisted circular RNA (racRNA) expression generates significantly higher levels of circular RNA than the previous circular RNA expressing system. HEK293T cells expressed racRNA Broccoli from a variety of racRNA expression systems (see Fig. 1 ) with different combinations of 5’ and 3’ ribozymes and were compared to expression using the tricY system. Cells were treated with actinomycin D (ActD) for 6 h to observe the drop in RNA levels after new RNA synthesis was inhibited. The Twister P1 and Twister P3 U2A construct, dubbed “Tornado”, expresses high levels of Broccoli RNA that exhibit high stability, characteristic of circRNA. d , Tornado-expressed RNA is decisively circular. DNA-directed cleavage by RNase H of a linear RNA produces two bands, each of expected size given the transcript length and probe site. The identical treatment of the same sequence expressed from Tornado produces a single band similar in size to the uncleaved transcribed sample.

    Techniques Used: Expressing, Construct, In Vitro, Polyacrylamide Gel Electrophoresis, Ligation, Staining, Fluorescence, Sequencing

    4) Product Images from "Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts"

    Article Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts

    Journal: Nature biotechnology

    doi: 10.1038/s41587-019-0090-6

    Tornado expression system generates circular RNA a , Ribozymes efficiently self-cleave during transcription reactions.. The construct containing Twister P1 and Twister P3 U2A ribozymes was transcribed in vitro and quenched with urea before running on denaturing PAGE and visualizing RNA. Fully cleaved products and the side products of cleavage accumulate efficiently and rapidly after transcription. b , Fully-cleaved products of transcription in a contain appropriate ends for circularization by the endogenous ligase, RtcB. We excised the fully-cleaved RNA from a and performed an RtcB ligation reactions. RtcB treatment produces a shift in gel mobility that is not observed without ligation or with pre-treatment with T4 PNK. This shift in gel mobility suggests that the fully-cleaved RNA contains the appropriate ends for ligation. Staining of the gel with DFHBI-1T and comparison of fluorescence relative to SYBR Gold signal demonstrates that circular Broccoli is brighter than linear Broccoli. c , Twister-based ribozyme-assisted circular RNA (racRNA) expression generates significantly higher levels of circular RNA than the previous circular RNA expressing system. HEK293T cells expressed racRNA Broccoli from a variety of racRNA expression systems (see Fig. 1 ) with different combinations of 5’ and 3’ ribozymes and were compared to expression using the tricY system. Cells were treated with actinomycin D (ActD) for 6 h to observe the drop in RNA levels after new RNA synthesis was inhibited. The Twister P1 and Twister P3 U2A construct, dubbed “Tornado”, expresses high levels of Broccoli RNA that exhibit high stability, characteristic of circRNA. d , Tornado-expressed RNA is decisively circular. DNA-directed cleavage by RNase H of a linear RNA produces two bands, each of expected size given the transcript length and probe site. The identical treatment of the same sequence expressed from Tornado produces a single band similar in size to the uncleaved transcribed sample.
    Figure Legend Snippet: Tornado expression system generates circular RNA a , Ribozymes efficiently self-cleave during transcription reactions.. The construct containing Twister P1 and Twister P3 U2A ribozymes was transcribed in vitro and quenched with urea before running on denaturing PAGE and visualizing RNA. Fully cleaved products and the side products of cleavage accumulate efficiently and rapidly after transcription. b , Fully-cleaved products of transcription in a contain appropriate ends for circularization by the endogenous ligase, RtcB. We excised the fully-cleaved RNA from a and performed an RtcB ligation reactions. RtcB treatment produces a shift in gel mobility that is not observed without ligation or with pre-treatment with T4 PNK. This shift in gel mobility suggests that the fully-cleaved RNA contains the appropriate ends for ligation. Staining of the gel with DFHBI-1T and comparison of fluorescence relative to SYBR Gold signal demonstrates that circular Broccoli is brighter than linear Broccoli. c , Twister-based ribozyme-assisted circular RNA (racRNA) expression generates significantly higher levels of circular RNA than the previous circular RNA expressing system. HEK293T cells expressed racRNA Broccoli from a variety of racRNA expression systems (see Fig. 1 ) with different combinations of 5’ and 3’ ribozymes and were compared to expression using the tricY system. Cells were treated with actinomycin D (ActD) for 6 h to observe the drop in RNA levels after new RNA synthesis was inhibited. The Twister P1 and Twister P3 U2A construct, dubbed “Tornado”, expresses high levels of Broccoli RNA that exhibit high stability, characteristic of circRNA. d , Tornado-expressed RNA is decisively circular. DNA-directed cleavage by RNase H of a linear RNA produces two bands, each of expected size given the transcript length and probe site. The identical treatment of the same sequence expressed from Tornado produces a single band similar in size to the uncleaved transcribed sample.

    Techniques Used: Expressing, Construct, In Vitro, Polyacrylamide Gel Electrophoresis, Ligation, Staining, Fluorescence, Sequencing

    Related Articles

    Incubation:

    Article Title: Small circRNAs with self-cleaving ribozymes are highly expressed in diverse metazoan transcriptomes
    Article Snippet: .. For the assays of RtcB ligation and self-ligation, 1–10 ng of gel-purified radiolabelled retrozyme RNAs, or 1–4 μg for non-radiolabelled RNAs, were firstly denatured at 95°C for 1 min, cooled down to 25°C (either 0.5°C/s or 1°C/min, with similar results), and then incubated either in the presence of RtcB ligase in its corresponding buffer for 1 h at 37°C (New England Biolabs) or in 50 mM Tris–HCl, pH 8 and 10 to 50 mM MgCl2 for 1 h at 25°C, respectively. ..

    Ligation:

    Article Title: Physiologic RNA Targets and Refined Sequence Specificity of Coronavirus EndoU
    Article Snippet: .. The ligation reactions were conducted using 15 pmol of RtcB ligase (NEB), 1x RtcB buffer (NEB), 100 μM GTP, 1 mM MnCl2 , 20 units of RNase inhibitor (Enzymatics) at 37°C for 2 h. Samples were ethanol precipitated with 20 μg of glycogen and resuspended in 10 μl of RNase free H2 O for chemical fragmentation (Ambion Fragmentation Reagent) at 65°C for 4 min. ..

    Article Title: Small circRNAs with self-cleaving ribozymes are highly expressed in diverse metazoan transcriptomes
    Article Snippet: .. For the assays of RtcB ligation and self-ligation, 1–10 ng of gel-purified radiolabelled retrozyme RNAs, or 1–4 μg for non-radiolabelled RNAs, were firstly denatured at 95°C for 1 min, cooled down to 25°C (either 0.5°C/s or 1°C/min, with similar results), and then incubated either in the presence of RtcB ligase in its corresponding buffer for 1 h at 37°C (New England Biolabs) or in 50 mM Tris–HCl, pH 8 and 10 to 50 mM MgCl2 for 1 h at 25°C, respectively. ..

    Article Title: Small circRNAs with self-cleaving ribozymes are frequently expressed in metazoan transcriptomes
    Article Snippet: .. For the assays of RtcB ligation, 1 to 10 ng of gel-purified retrozyme RNA were ligated using RtcB ligase in its corresponding buffer for 1 h at 37 °C (New England Biolabs). .. Best results for self-ligation assays were obtained with 1 to 10 ng of gel-purified retrozyme RNA in 50 mM Tris–HCl, pH 8, which were firstly denatured at 95 °C for 1 min, slowly cooled down to 25 °C (1 °C decrease every 2 seconds), and then incubated in the presence of 10 to 50 mM MgCl2 for 1h at 25 °C.

    Purification:

    Article Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts
    Article Snippet: .. 10 pmol of this purified T4-PNK-treated RNA or of the gel purified RNA was ligated using RtcB Ligase (New England Biolabs M0458) for 1 h at 37 °C. ..

    Article Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts
    Article Snippet: .. 10 pmol of this purified T4-PNK-treated RNA or of the gel purified RNA was ligated using RtcB Ligase (New England Biolabs M0458) for 1 h at 37 °C. .. Cloning of autocatalytic circRNA constructs DNA templates containing Broccoli and each of the ribozyme combinations were prepared as described above with flanking SalI and XbaI restriction sites.

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  • 95
    New England Biolabs rtcb ligase
    Circularization of Mexican axolotl self-cleaved <t>retrozyme</t> RNA. Purified linear monomeric RNA resulting from double self-cleavage of DRtz353 (see Figure 4C ) was run directly in a denaturing gel (lane 1), after 1h incubation with <t>RtcB</t> ligase in its corresponding buffer (lane 2), in RtcB buffer without protein ligase (lane 3), and in 50 mM Mg 2+ buffer (lane 4). Circular RNAs can be readily detected in lane 2 (up to 80% circularization for the 350 nt monomer) and lane 4 (3% self-circularization for the 350 nt monomer). Minor fractions of linear and circular dimeric RNAs are also indicated (lane 2).
    Rtcb Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rtcb ligase/product/New England Biolabs
    Average 95 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    rtcb ligase - by Bioz Stars, 2020-08
    95/100 stars
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    Circularization of Mexican axolotl self-cleaved retrozyme RNA. Purified linear monomeric RNA resulting from double self-cleavage of DRtz353 (see Figure 4C ) was run directly in a denaturing gel (lane 1), after 1h incubation with RtcB ligase in its corresponding buffer (lane 2), in RtcB buffer without protein ligase (lane 3), and in 50 mM Mg 2+ buffer (lane 4). Circular RNAs can be readily detected in lane 2 (up to 80% circularization for the 350 nt monomer) and lane 4 (3% self-circularization for the 350 nt monomer). Minor fractions of linear and circular dimeric RNAs are also indicated (lane 2).

    Journal: bioRxiv

    Article Title: Small circRNAs with self-cleaving ribozymes are frequently expressed in metazoan transcriptomes

    doi: 10.1101/721605

    Figure Lengend Snippet: Circularization of Mexican axolotl self-cleaved retrozyme RNA. Purified linear monomeric RNA resulting from double self-cleavage of DRtz353 (see Figure 4C ) was run directly in a denaturing gel (lane 1), after 1h incubation with RtcB ligase in its corresponding buffer (lane 2), in RtcB buffer without protein ligase (lane 3), and in 50 mM Mg 2+ buffer (lane 4). Circular RNAs can be readily detected in lane 2 (up to 80% circularization for the 350 nt monomer) and lane 4 (3% self-circularization for the 350 nt monomer). Minor fractions of linear and circular dimeric RNAs are also indicated (lane 2).

    Article Snippet: For the assays of RtcB ligation, 1 to 10 ng of gel-purified retrozyme RNA were ligated using RtcB ligase in its corresponding buffer for 1 h at 37 °C (New England Biolabs).

    Techniques: Purification, Incubation

    Tornado expression system generates circular RNA a , Ribozymes efficiently self-cleave during transcription reactions.. The construct containing Twister P1 and Twister P3 U2A ribozymes was transcribed in vitro and quenched with urea before running on denaturing PAGE and visualizing RNA. Fully cleaved products and the side products of cleavage accumulate efficiently and rapidly after transcription. b , Fully-cleaved products of transcription in a contain appropriate ends for circularization by the endogenous ligase, RtcB. We excised the fully-cleaved RNA from a and performed an RtcB ligation reactions. RtcB treatment produces a shift in gel mobility that is not observed without ligation or with pre-treatment with T4 PNK. This shift in gel mobility suggests that the fully-cleaved RNA contains the appropriate ends for ligation. Staining of the gel with DFHBI-1T and comparison of fluorescence relative to SYBR Gold signal demonstrates that circular Broccoli is brighter than linear Broccoli. c , Twister-based ribozyme-assisted circular RNA (racRNA) expression generates significantly higher levels of circular RNA than the previous circular RNA expressing system. HEK293T cells expressed racRNA Broccoli from a variety of racRNA expression systems (see Fig. 1 ) with different combinations of 5’ and 3’ ribozymes and were compared to expression using the tricY system. Cells were treated with actinomycin D (ActD) for 6 h to observe the drop in RNA levels after new RNA synthesis was inhibited. The Twister P1 and Twister P3 U2A construct, dubbed “Tornado”, expresses high levels of Broccoli RNA that exhibit high stability, characteristic of circRNA. d , Tornado-expressed RNA is decisively circular. DNA-directed cleavage by RNase H of a linear RNA produces two bands, each of expected size given the transcript length and probe site. The identical treatment of the same sequence expressed from Tornado produces a single band similar in size to the uncleaved transcribed sample.

    Journal: Nature biotechnology

    Article Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts

    doi: 10.1038/s41587-019-0090-6

    Figure Lengend Snippet: Tornado expression system generates circular RNA a , Ribozymes efficiently self-cleave during transcription reactions.. The construct containing Twister P1 and Twister P3 U2A ribozymes was transcribed in vitro and quenched with urea before running on denaturing PAGE and visualizing RNA. Fully cleaved products and the side products of cleavage accumulate efficiently and rapidly after transcription. b , Fully-cleaved products of transcription in a contain appropriate ends for circularization by the endogenous ligase, RtcB. We excised the fully-cleaved RNA from a and performed an RtcB ligation reactions. RtcB treatment produces a shift in gel mobility that is not observed without ligation or with pre-treatment with T4 PNK. This shift in gel mobility suggests that the fully-cleaved RNA contains the appropriate ends for ligation. Staining of the gel with DFHBI-1T and comparison of fluorescence relative to SYBR Gold signal demonstrates that circular Broccoli is brighter than linear Broccoli. c , Twister-based ribozyme-assisted circular RNA (racRNA) expression generates significantly higher levels of circular RNA than the previous circular RNA expressing system. HEK293T cells expressed racRNA Broccoli from a variety of racRNA expression systems (see Fig. 1 ) with different combinations of 5’ and 3’ ribozymes and were compared to expression using the tricY system. Cells were treated with actinomycin D (ActD) for 6 h to observe the drop in RNA levels after new RNA synthesis was inhibited. The Twister P1 and Twister P3 U2A construct, dubbed “Tornado”, expresses high levels of Broccoli RNA that exhibit high stability, characteristic of circRNA. d , Tornado-expressed RNA is decisively circular. DNA-directed cleavage by RNase H of a linear RNA produces two bands, each of expected size given the transcript length and probe site. The identical treatment of the same sequence expressed from Tornado produces a single band similar in size to the uncleaved transcribed sample.

    Article Snippet: 10 pmol of this purified T4-PNK-treated RNA or of the gel purified RNA was ligated using RtcB Ligase (New England Biolabs M0458) for 1 h at 37 °C.

    Techniques: Expressing, Construct, In Vitro, Polyacrylamide Gel Electrophoresis, Ligation, Staining, Fluorescence, Sequencing