t4 rna ligase 2  (New England Biolabs)


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    New England Biolabs t4 rna ligase 2
    T4 Rna Ligase 2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 1 article reviews
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    t4 rna ligase 2 - by Bioz Stars, 2020-05
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    Random Hexamer Labeling:

    Article Title: Spliceosome profiling visualizes operations of a dynamic RNP at nucleotide resolution
    Article Snippet: .. “A” sample RNA was ligated to a pre-adenylated adaptor with a 5′ random hexamer (see ) using T4 RNA ligase 2, truncated, K227Q (NEB) for 2.5 hours at 37°C (25% PEG-8000, 12.5% DMSO, 1X buffer). .. Hydrolyzed RNA ends were then repaired by treating with 25 units PNK (NEB) for 1 hour at 37°C.

    other:

    Article Title: Protocol for Ribosome Profiling in Bacteria
    Article Snippet: Add 1 μl of T4 RNA ligase 2 truncated to each sample.

    Incubation:

    Article Title: NAD captureSeq indicates NAD as a bacterial cap for a subset of regulatory RNAs.
    Article Snippet: .. A distinctive feature of prokaryotic gene expression is the absence of 5'-capped RNA. .. A distinctive feature of prokaryotic gene expression is the absence of 5'-capped RNA.

    Ligation:

    Article Title: NAD captureSeq indicates NAD as a bacterial cap for a subset of regulatory RNAs.
    Article Snippet: .. A distinctive feature of prokaryotic gene expression is the absence of 5'-capped RNA. .. A distinctive feature of prokaryotic gene expression is the absence of 5'-capped RNA.

    Article Title: The Drosophila RNA Helicase Belle (DDX3) Non-Autonomously Suppresses Germline Tumorigenesis Via Regulation of a Specific mRNA Set
    Article Snippet: .. A previously pre-adenylated 3′-adapter (5′DNA Adenylation Kit (NEB) was used for preparation) was ligated with RNAs on-bead by T4 RNA ligase 2 (NEB) 400 U in 30 µL of ligation reaction 2 h at 25°C on a thermal mixer. ..

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    New England Biolabs t4 rna ligase 2
    Graphical Visualization of the 3′ RACE-Seq Approach, Related to Figure 2 (A) Graphical representation of 3′ RACE-seq library preparation and the oligonucleotides used. First, the 3′ adaptor RA3_15N was joined to the 3′ end of RNA by enzymatic ligation. The adaptor has: (i) 5′ rApp modification for efficient and specific ligation by the truncated <t>T4</t> RNA ligase 2, (ii) delimiter sequence to be used in bioinformatics analyses to exclude RT and PCR artifacts (CTGAC, highlighted in violet), (iii) unique 15N barcode for individual transcript barcoding (highlighted in green), (iv) anchor sequence to pair with the reverse transcription primer (underlined) and (v) dideoxyC on the 3′ end to prevent concatamer formation. The RNA ligated to the adaptor sequence was purified from excess adaptor and reverse transcription was performed with the RT primer, which is compatible with Illumina sequencing and has: (i) sequences to base-pair with the adaptor (underlined), (ii) 6-nucleotide barcode for sample barcoding (highlighted in red), (iii) sequences that base pair with the universal outer primer for nested PCR (blue). Libraries were generated by nested PCR with 2 outer forward primers (F1 and F2) and a single universal reverse primer (uni rev). PCR amplicons of first and second PCRs were purified from excess primers on AmPure beads (Agencourt) before beginning the next step. (B) Flowchart of the bioinformatics approach to 3′ RACE-seq data analysis. The procedure starts at the top. Datasets are shown in rectangles. Software used is depicted in hexagons.
    T4 Rna Ligase 2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase 2 - by Bioz Stars, 2020-05
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    99
    New England Biolabs t4 rna ligase
    The RNA-ligase-mediated 5′-RACE procedure to clone the 5′ terminus of a viral RNA genome. Viral RNA is converted to cDNA with random primers by reverse transcription. A phosphorylated synthetic oligodeoxynucleotide adapter is ligated to the 3′ end of cDNA by using <t>T4</t> RNA ligase, and then two rounds of PCR amplification are performed. The first-round PCR is done with the adapter primer (AP-1), complementary to the 3′ end of the adapter, and the viral-specific primer 1 (VS-1). The second-round nested PCR is done with the other adapter primer (AP-2), complementary to the 5′ portion of the adapter, and the viral-specific primer 2 (VS-2). The PCR products are subjected to cloning and then sequencing. The RNA molecule is represented as a wavy line, cDNA molecules are straight lines, adapters are thick lines, and primers are short arrows.
    T4 Rna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
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    Graphical Visualization of the 3′ RACE-Seq Approach, Related to Figure 2 (A) Graphical representation of 3′ RACE-seq library preparation and the oligonucleotides used. First, the 3′ adaptor RA3_15N was joined to the 3′ end of RNA by enzymatic ligation. The adaptor has: (i) 5′ rApp modification for efficient and specific ligation by the truncated T4 RNA ligase 2, (ii) delimiter sequence to be used in bioinformatics analyses to exclude RT and PCR artifacts (CTGAC, highlighted in violet), (iii) unique 15N barcode for individual transcript barcoding (highlighted in green), (iv) anchor sequence to pair with the reverse transcription primer (underlined) and (v) dideoxyC on the 3′ end to prevent concatamer formation. The RNA ligated to the adaptor sequence was purified from excess adaptor and reverse transcription was performed with the RT primer, which is compatible with Illumina sequencing and has: (i) sequences to base-pair with the adaptor (underlined), (ii) 6-nucleotide barcode for sample barcoding (highlighted in red), (iii) sequences that base pair with the universal outer primer for nested PCR (blue). Libraries were generated by nested PCR with 2 outer forward primers (F1 and F2) and a single universal reverse primer (uni rev). PCR amplicons of first and second PCRs were purified from excess primers on AmPure beads (Agencourt) before beginning the next step. (B) Flowchart of the bioinformatics approach to 3′ RACE-seq data analysis. The procedure starts at the top. Datasets are shown in rectangles. Software used is depicted in hexagons.

    Journal: Cell

    Article Title: Uridylation by TUT4/7 Restricts Retrotransposition of Human LINE-1s

    doi: 10.1016/j.cell.2018.07.022

    Figure Lengend Snippet: Graphical Visualization of the 3′ RACE-Seq Approach, Related to Figure 2 (A) Graphical representation of 3′ RACE-seq library preparation and the oligonucleotides used. First, the 3′ adaptor RA3_15N was joined to the 3′ end of RNA by enzymatic ligation. The adaptor has: (i) 5′ rApp modification for efficient and specific ligation by the truncated T4 RNA ligase 2, (ii) delimiter sequence to be used in bioinformatics analyses to exclude RT and PCR artifacts (CTGAC, highlighted in violet), (iii) unique 15N barcode for individual transcript barcoding (highlighted in green), (iv) anchor sequence to pair with the reverse transcription primer (underlined) and (v) dideoxyC on the 3′ end to prevent concatamer formation. The RNA ligated to the adaptor sequence was purified from excess adaptor and reverse transcription was performed with the RT primer, which is compatible with Illumina sequencing and has: (i) sequences to base-pair with the adaptor (underlined), (ii) 6-nucleotide barcode for sample barcoding (highlighted in red), (iii) sequences that base pair with the universal outer primer for nested PCR (blue). Libraries were generated by nested PCR with 2 outer forward primers (F1 and F2) and a single universal reverse primer (uni rev). PCR amplicons of first and second PCRs were purified from excess primers on AmPure beads (Agencourt) before beginning the next step. (B) Flowchart of the bioinformatics approach to 3′ RACE-seq data analysis. The procedure starts at the top. Datasets are shown in rectangles. Software used is depicted in hexagons.

    Article Snippet: The reactions were carried out in 20 μL with 1x T4 RNA ligase 2 truncated buffer (NEB) supplemented with PEG-8000 at 10% final concentration, 0.25 U/μl RiboLock inhibitor (Thermo Fisher Scientific), 3 pmol of the 5′ FAM-labeled 44-mer oligonucleotide RNA44 (Future Synthesis) and 300 U T4 RNA ligase 2 truncated (NEB) for 18h at 18°C.

    Techniques: Ligation, Modification, Sequencing, Polymerase Chain Reaction, Purification, Nested PCR, Generated, Software

    The RNA-ligase-mediated 5′-RACE procedure to clone the 5′ terminus of a viral RNA genome. Viral RNA is converted to cDNA with random primers by reverse transcription. A phosphorylated synthetic oligodeoxynucleotide adapter is ligated to the 3′ end of cDNA by using T4 RNA ligase, and then two rounds of PCR amplification are performed. The first-round PCR is done with the adapter primer (AP-1), complementary to the 3′ end of the adapter, and the viral-specific primer 1 (VS-1). The second-round nested PCR is done with the other adapter primer (AP-2), complementary to the 5′ portion of the adapter, and the viral-specific primer 2 (VS-2). The PCR products are subjected to cloning and then sequencing. The RNA molecule is represented as a wavy line, cDNA molecules are straight lines, adapters are thick lines, and primers are short arrows.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Cloning and characterization of the extreme 5?-terminal sequences of the RNA genomes of GB virus C/hepatitis G virus

    doi:

    Figure Lengend Snippet: The RNA-ligase-mediated 5′-RACE procedure to clone the 5′ terminus of a viral RNA genome. Viral RNA is converted to cDNA with random primers by reverse transcription. A phosphorylated synthetic oligodeoxynucleotide adapter is ligated to the 3′ end of cDNA by using T4 RNA ligase, and then two rounds of PCR amplification are performed. The first-round PCR is done with the adapter primer (AP-1), complementary to the 3′ end of the adapter, and the viral-specific primer 1 (VS-1). The second-round nested PCR is done with the other adapter primer (AP-2), complementary to the 5′ portion of the adapter, and the viral-specific primer 2 (VS-2). The PCR products are subjected to cloning and then sequencing. The RNA molecule is represented as a wavy line, cDNA molecules are straight lines, adapters are thick lines, and primers are short arrows.

    Article Snippet: The ligation solution contained 5 pg of the synthetic oligonucleotide adapter, 50 mM Tris·HCl (pH 7.8), 10 mM MgCl2 , 1 mM 2-mercaptoethanol, 1 mM ATP, 20 units of human placenta ribonuclease inhibitor (RNasin, Promega), and 20 units of T4 RNA ligase (New England BioLabs).

    Techniques: Polymerase Chain Reaction, Amplification, Nested PCR, Clone Assay, Sequencing

    Main cleavage sites in the 25S rRNA are located in loop regions. ( A ) Northern hybridizations of total yeast RNA extracted from wild-type W303 cells treated with different concentrations of H 2 O 2 . Hybridizations with probes against 25S (probe 007, position +40, lanes 1–6; probe W234, position +344, lanes 7–12; probe W236, position +600, lanes 13–18; probe W238, position +843, lanes 19–24; probe W239, position +2168, lanes 25–30 and probe W240, position +3323, lanes 31–36). Asterisks above the arrows indicate the products that were further analysed. Arrow marked with a hatch shows a band matching the potential 3′ product of the major cleavage, 5′ product is marked with one asterisk. ( B–C ) Primer extension analysis for two main cleavage sites in the 25S rRNA in W303 cells treated with 1 mM H 2 O 2 (A) and in 16-day old chronologically aged rho0 W303 cells (B). Primer extensions were performed using primers W235 for sites around positions +400 and +470 and W237 for sites around position +600 relative to the 5′ end of the mature 25S. DNA sequencing on a PCR product encompassing the 5′ end of the 25S from +40 to +701, using the same primers was run in parallel on 6% sequencing polyacrylamide gels (lanes 1–4). The sequences with primer extension stops are shown on the right. Secondary structures of the regions in the vicinity of the cleavages, indicated by arrowheads and shown beside corresponding primer extension reactions, were adapted from the website http://rna.icmb.utexas.edu/ . ( D–E ) 3′ ends of cleaved-off products for the major cleavage at positions +610–611 were mapped by 3′ RACE. (D) PCR reactions on cDNA prepared using total RNA from untreated control (lane 1, C) and cells treated with 1 mM H 2 O 2 (lane 2). To generate cDNA, total RNA that had been ligated to an ‘anchor’ oligonucleotide (W242) with T4 RNA ligase, was reverse transcribed using a primer specific for the anchor (W243). This was followed by PCR reaction using the same 3′ primer and the 5′ primer starting at position +50 in the 25S rRNA (W241). Arrows indicate products corresponding to fragments cleaved at +398–404 (lower) and +610–611 (upper). PCR fragments were cloned into pGEM-Teasy and sequenced. (E) Sequences obtained by the 3′ RACE analysis for fragments cleaved at site +398–403 (19 independent clones) and site +610–611 (10 independent clones). The corresponding regions of the 25S with cleavage sites mapped by primer extension and indicated with empty arrowheads are shown above in grey. Figures in parentheses show the number of identical clones. ( F ) Mapping 3′ ends of two major cleavages sites using RNase H cleavage on total RNA extracted from wild-type, rrp41-1 and ski7Δ cells treated with 1mM H 2 O 2 (lanes, 2–4) and from wild-type untreated control (lane 1, C). RNase H treatment was performed on RNA samples annealed to DNA oligonucleotides W244 and W263 complementary to positions +271 and +510, respectively. Samples were separated on a 8% acrylamide gel and hybridized with probe W234 (F-I) and probe W264 (F-II) to detect 3′ ends of fragments cleaved at +398–403 (F-I) and at +610–611 (F-II), respectively. Arrows show more defined 3′ ends of products cleaved at +610–611 for all strains and at +398–403 in the mutants; vertical bar in F-I indicates heterogenous 3′ ends of products cleaved at +398–403 in wild-type cells.

    Journal: Nucleic Acids Research

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast

    doi: 10.1093/nar/gkm1100

    Figure Lengend Snippet: Main cleavage sites in the 25S rRNA are located in loop regions. ( A ) Northern hybridizations of total yeast RNA extracted from wild-type W303 cells treated with different concentrations of H 2 O 2 . Hybridizations with probes against 25S (probe 007, position +40, lanes 1–6; probe W234, position +344, lanes 7–12; probe W236, position +600, lanes 13–18; probe W238, position +843, lanes 19–24; probe W239, position +2168, lanes 25–30 and probe W240, position +3323, lanes 31–36). Asterisks above the arrows indicate the products that were further analysed. Arrow marked with a hatch shows a band matching the potential 3′ product of the major cleavage, 5′ product is marked with one asterisk. ( B–C ) Primer extension analysis for two main cleavage sites in the 25S rRNA in W303 cells treated with 1 mM H 2 O 2 (A) and in 16-day old chronologically aged rho0 W303 cells (B). Primer extensions were performed using primers W235 for sites around positions +400 and +470 and W237 for sites around position +600 relative to the 5′ end of the mature 25S. DNA sequencing on a PCR product encompassing the 5′ end of the 25S from +40 to +701, using the same primers was run in parallel on 6% sequencing polyacrylamide gels (lanes 1–4). The sequences with primer extension stops are shown on the right. Secondary structures of the regions in the vicinity of the cleavages, indicated by arrowheads and shown beside corresponding primer extension reactions, were adapted from the website http://rna.icmb.utexas.edu/ . ( D–E ) 3′ ends of cleaved-off products for the major cleavage at positions +610–611 were mapped by 3′ RACE. (D) PCR reactions on cDNA prepared using total RNA from untreated control (lane 1, C) and cells treated with 1 mM H 2 O 2 (lane 2). To generate cDNA, total RNA that had been ligated to an ‘anchor’ oligonucleotide (W242) with T4 RNA ligase, was reverse transcribed using a primer specific for the anchor (W243). This was followed by PCR reaction using the same 3′ primer and the 5′ primer starting at position +50 in the 25S rRNA (W241). Arrows indicate products corresponding to fragments cleaved at +398–404 (lower) and +610–611 (upper). PCR fragments were cloned into pGEM-Teasy and sequenced. (E) Sequences obtained by the 3′ RACE analysis for fragments cleaved at site +398–403 (19 independent clones) and site +610–611 (10 independent clones). The corresponding regions of the 25S with cleavage sites mapped by primer extension and indicated with empty arrowheads are shown above in grey. Figures in parentheses show the number of identical clones. ( F ) Mapping 3′ ends of two major cleavages sites using RNase H cleavage on total RNA extracted from wild-type, rrp41-1 and ski7Δ cells treated with 1mM H 2 O 2 (lanes, 2–4) and from wild-type untreated control (lane 1, C). RNase H treatment was performed on RNA samples annealed to DNA oligonucleotides W244 and W263 complementary to positions +271 and +510, respectively. Samples were separated on a 8% acrylamide gel and hybridized with probe W234 (F-I) and probe W264 (F-II) to detect 3′ ends of fragments cleaved at +398–403 (F-I) and at +610–611 (F-II), respectively. Arrows show more defined 3′ ends of products cleaved at +610–611 for all strains and at +398–403 in the mutants; vertical bar in F-I indicates heterogenous 3′ ends of products cleaved at +398–403 in wild-type cells.

    Article Snippet: To this end, DNA ‘anchor’ oligonucleotide (W242) was ligated with T4 RNA ligase to total RNA from untreated and treated W303 cells to prepare cDNA using a primer specific for the anchor (W243).

    Techniques: Northern Blot, DNA Sequencing, Polymerase Chain Reaction, Sequencing, Clone Assay, Acrylamide Gel Assay

    RNase H cleavage (Step 2) and direct non-splinted cross-religation using T4 RNA ligase (Step 3). ( a ) Denaturing anion-exchange HPLC profile of fragments obtained by site-specific RNase H cleavage in SL2 between A29 and C30 of the RsmZ RNA (60 nmol reaction). The RNase H cleavage was performed with a chimera/RNA ratio of 0.75:1. The different fragments obtained by RNase H cleavage are shown on the top of their corresponding peak. Side-products occurring because of ‘unspecific’ cleavage in SL4 are marked by asterisks (see Supplementary Figure S3 ). The retention time of the HPLC profile is indicated on the y-axis. The purification conditions used are presented in the methods section. ( b ) Scheme of RNase H cleavage reaction and corresponding reaction yields. The yield of the cleavage reaction before HPLC purification is indicated, the values in brackets are expressing the yield after purification. The site of cleavage is shown by scissors. ( c ) Analytical 16% denaturing PAGE gel of the ligation reaction. Left lane: 400 pmol of each 5′-RNA (29 nt) and 3′-RNA (43 nt) before ligation, right lane: after ligation. ( d ) Reaction scheme and corresponding reaction yields for T4 RNA ligase mediated non-splinted cross-ligation of both a labeled (in red) and an unlabeled (in black) fragment, respectively. The ligation yield determined with a reaction using only unlabeled fragments is indicated.

    Journal: Nucleic Acids Research

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage

    doi: 10.1093/nar/gkq756

    Figure Lengend Snippet: RNase H cleavage (Step 2) and direct non-splinted cross-religation using T4 RNA ligase (Step 3). ( a ) Denaturing anion-exchange HPLC profile of fragments obtained by site-specific RNase H cleavage in SL2 between A29 and C30 of the RsmZ RNA (60 nmol reaction). The RNase H cleavage was performed with a chimera/RNA ratio of 0.75:1. The different fragments obtained by RNase H cleavage are shown on the top of their corresponding peak. Side-products occurring because of ‘unspecific’ cleavage in SL4 are marked by asterisks (see Supplementary Figure S3 ). The retention time of the HPLC profile is indicated on the y-axis. The purification conditions used are presented in the methods section. ( b ) Scheme of RNase H cleavage reaction and corresponding reaction yields. The yield of the cleavage reaction before HPLC purification is indicated, the values in brackets are expressing the yield after purification. The site of cleavage is shown by scissors. ( c ) Analytical 16% denaturing PAGE gel of the ligation reaction. Left lane: 400 pmol of each 5′-RNA (29 nt) and 3′-RNA (43 nt) before ligation, right lane: after ligation. ( d ) Reaction scheme and corresponding reaction yields for T4 RNA ligase mediated non-splinted cross-ligation of both a labeled (in red) and an unlabeled (in black) fragment, respectively. The ligation yield determined with a reaction using only unlabeled fragments is indicated.

    Article Snippet: A typical large-scale ligation reaction using T4 RNA ligase was 40 μM in both RNA fragments in 1× NEB ligation buffer (50 mM Tris–HCl pH = 7.8, 1 mM ATP, 10 mM MgCl2 , 10 mM DTT), 1x in BSA using 5 U T4 RNA ligase per nmol of RNA to be ligated.

    Techniques: High Performance Liquid Chromatography, Purification, Expressing, Polyacrylamide Gel Electrophoresis, Ligation, Labeling