t4 rnl2  (New England Biolabs)


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    Name:
    T4 RNA Ligase 2
    Description:
    T4 RNA Ligase 2 750 units
    Catalog Number:
    m0239l
    Price:
    312
    Size:
    750 units
    Category:
    RNA Ligases
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    New England Biolabs t4 rnl2
    T4 RNA Ligase 2
    T4 RNA Ligase 2 750 units
    https://www.bioz.com/result/t4 rnl2/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t4 rnl2 - by Bioz Stars, 2020-02
    95/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast
    Article Snippet: DNA ‘adaptor’ oligonucleotide (W242) carrying aminolinker at the 3′-end was ligated with the 3′-end of total RNA using 20U of T4 RNA ligase (NEB). .. RNA was extracted with phenol: chloroform: isoamyl alcohol (v/v 25:24:1), precipitated and used as a template for cDNA synthesis using W243 primer complementary to the anchor sequence and the Enhanced Avian HS RT-PCR Kit (Sigma) according to manufacturer's instructions. cDNA was amplified using primers W241 and W243, the resulting PCR product was gel purified, cloned into pGEM-T Easy vector and sequenced using primer W241.

    Article Title: Cloning and characterization of the extreme 5?-terminal sequences of the RNA genomes of GB virus C/hepatitis G virus
    Article Snippet: Paragraph title: Cloning of the 5′-Terminal Sequences of the Hepatitis GBV-C/HGV Viral Genome. ... 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).

    Amplification:

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast
    Article Snippet: DNA ‘adaptor’ oligonucleotide (W242) carrying aminolinker at the 3′-end was ligated with the 3′-end of total RNA using 20U of T4 RNA ligase (NEB). .. RNA was extracted with phenol: chloroform: isoamyl alcohol (v/v 25:24:1), precipitated and used as a template for cDNA synthesis using W243 primer complementary to the anchor sequence and the Enhanced Avian HS RT-PCR Kit (Sigma) according to manufacturer's instructions. cDNA was amplified using primers W241 and W243, the resulting PCR product was gel purified, cloned into pGEM-T Easy vector and sequenced using primer W241.

    Polymerase Chain Reaction:

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast
    Article Snippet: Dideoxy-DNA sequencing was performed on PCR-templates prepared from genomic yeast DNA using the same primers as for primer extension (W235 and W237) and a fMol Seq kit (Promega) according to manufacturer's instructions. .. DNA ‘adaptor’ oligonucleotide (W242) carrying aminolinker at the 3′-end was ligated with the 3′-end of total RNA using 20U of T4 RNA ligase (NEB).

    Article Title: Cloning and characterization of the extreme 5?-terminal sequences of the RNA genomes of GB virus C/hepatitis G virus
    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). .. One microliter of the cDNA was then subjected to a first-round of PCR with an adapter primer (AP-1) complementary to the 3′ region of the adapter and an outer viral-specific primer (VS-1).

    Blocking Assay:

    Article Title: Blocking of targeted microRNAs from next-generation sequencing libraries
    Article Snippet: Paragraph title: Blocking hsa-miR-16–5p in sequencing libraries ... Although truncated T4 RNA Ligase 2 cannot turnover ATP, ATP can still bind to the remnants of the active site, leading to inhibition of the enzyme (personal communication with NEB).

    Incubation:

    Article Title: A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage
    Article Snippet: .. The 5-nt RNA 5′-CUCUU-3′ (40 nmol) and phosphorylated dinucleotide 24 (pC2′-X G5′-Y ) (20 nmol) were incubated together with 200 U of T4 RNA ligase (NEB), 10 µl DMSO, and 1× T4 RNA ligase buffer (50 mM Tris–HCl, pH 7.78, 10 mM MgCl2 , 10 mM dithiothreitol, 1 mM ATP) in a 100 μl reaction volume at 37°C for 6 h. The reaction mixture was worked up by PCI and ether extraction of the aqueous layers. .. The ligated product was purified by high pressure liquid chromatography on a C18 reversed-phase column (Vydac 201SP54) (6% acetonitrile/94% 0.1 M triethylammonium acetate, pH 7.0, for 5 min; then 6–16% acetonitrile/94–84% 0.1 M triethylammonium acetate, pH 7.0, over 30 min) and evaporated to dryness before further use.

    Article Title: Simple and efficient synthesis of 5? pre-adenylated DNA using thermostable RNA ligase
    Article Snippet: .. Ten microliter ligation reactions containing 5 pmol of the RNA acceptor, 7 pmol AppDNA17c-NH2 in 10 mM Tris–HCl pH 7.5 buffer, 10 mM MgCl2 , 1 mM DTT and 200 U of truncated T4 RNA ligase 2 were incubated for 2 h at 25°C. .. Reactions were stopped by adding 5 µl formamide loading buffer, heat inactivated at 95°C for 3 min and the products were separated, stained and visualized as described for the DNA adenylation above.

    Article Title: Human tRNA-derived small RNAs in the global regulation of RNA silencing
    Article Snippet: Fifteen-microliter reactions were incubated with the indicated enzymes at 37°C (Terminator Exonuclease: 30°C) for 60 min, acid phenol/chloroform extracted, ethanol precipitated, and resuspended for the second round of enzyme treatments, which was again followed by acid phenol/chloroform extraction, ethanol precipitation, and resuspension in PAGE loading buffer for Northern blot. .. Amounts of enzymes used: 15 units (U) of T4 PNK, 3′ phophatase ± (NEB M0201/m0236); 8 U of Tobacco Acid Pyrophosphatase (Epicentre Biotechnologies); 3 U of Terminator Exonuclease (Epicentre Biotechnologies); 4 U polyA polymerase (PAP; Ambion); and 15 U T4 RNA ligase (NEB).

    Modification:

    Article Title: Cloning and characterization of the extreme 5?-terminal sequences of the RNA genomes of GB virus C/hepatitis G virus
    Article Snippet: We utilized a modified 5′-RACE procedure ( , ). .. 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).

    Hybridization:

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast
    Article Snippet: Oligonucletides used for RNA hybridization and primer extension (W235 and W237) are listed in Supplementary Table S2 . .. DNA ‘adaptor’ oligonucleotide (W242) carrying aminolinker at the 3′-end was ligated with the 3′-end of total RNA using 20U of T4 RNA ligase (NEB).

    High Performance Liquid Chromatography:

    Article Title: A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage
    Article Snippet: The phosphorylated dinucleotide ( ) was purified on a C18 reversed-phase high pressure liquid chromatography column (Vydac 201SP54) (0–30% acetonitrile/100–70% 0.1 M triethylammonium acetate, pH 7.0, over 30 min) and evaporated to dryness before further use. .. The 5-nt RNA 5′-CUCUU-3′ (40 nmol) and phosphorylated dinucleotide 24 (pC2′-X G5′-Y ) (20 nmol) were incubated together with 200 U of T4 RNA ligase (NEB), 10 µl DMSO, and 1× T4 RNA ligase buffer (50 mM Tris–HCl, pH 7.78, 10 mM MgCl2 , 10 mM dithiothreitol, 1 mM ATP) in a 100 μl reaction volume at 37°C for 6 h. The reaction mixture was worked up by PCI and ether extraction of the aqueous layers.

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage
    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. .. The reactions were subjected to HPLC purification followed by n -butanol extraction and lyophilization.

    Ligation:

    Article Title: A fully enzymatic method for site-directed spin labeling of long RNA
    Article Snippet: Paragraph title: Ligation of RNA segments ... The T4 RNA ligase 2 was purchased from New Englands Biolabs (United States).

    Article Title: A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage
    Article Snippet: Paragraph title: RNA ligase-mediated ligation to synthesize 5′-CUC UUC2′-X G5′-Y ... The 5-nt RNA 5′-CUCUU-3′ (40 nmol) and phosphorylated dinucleotide 24 (pC2′-X G5′-Y ) (20 nmol) were incubated together with 200 U of T4 RNA ligase (NEB), 10 µl DMSO, and 1× T4 RNA ligase buffer (50 mM Tris–HCl, pH 7.78, 10 mM MgCl2 , 10 mM dithiothreitol, 1 mM ATP) in a 100 μl reaction volume at 37°C for 6 h. The reaction mixture was worked up by PCI and ether extraction of the aqueous layers.

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast
    Article Snippet: DNA ‘adaptor’ oligonucleotide (W242) carrying aminolinker at the 3′-end was ligated with the 3′-end of total RNA using 20U of T4 RNA ligase (NEB). .. Ligation was performed in the presence of 25% PEG1000 (Sigma) at 37°C.

    Article Title: Simple and efficient synthesis of 5? pre-adenylated DNA using thermostable RNA ligase
    Article Snippet: .. Ten microliter ligation reactions containing 5 pmol of the RNA acceptor, 7 pmol AppDNA17c-NH2 in 10 mM Tris–HCl pH 7.5 buffer, 10 mM MgCl2 , 1 mM DTT and 200 U of truncated T4 RNA ligase 2 were incubated for 2 h at 25°C. .. Reactions were stopped by adding 5 µl formamide loading buffer, heat inactivated at 95°C for 3 min and the products were separated, stained and visualized as described for the DNA adenylation above.

    Article Title: Uridylation by TUT4/7 Restricts Retrotransposition of Human LINE-1s
    Article Snippet: RNA (∼2-3 μg) for each sample was used in a ligation reaction containing 125 pmol RA3_15N 3′ adaptor (5′ preadenylated and individually barcoded with a 15N sequence). .. 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.

    Article Title: Human tRNA-derived small RNAs in the global regulation of RNA silencing
    Article Snippet: Amounts of enzymes used: 15 units (U) of T4 PNK, 3′ phophatase ± (NEB M0201/m0236); 8 U of Tobacco Acid Pyrophosphatase (Epicentre Biotechnologies); 3 U of Terminator Exonuclease (Epicentre Biotechnologies); 4 U polyA polymerase (PAP; Ambion); and 15 U T4 RNA ligase (NEB). .. For 3′-adapter ligation with T4 RNA ligase, a noncommercial buffer without ATP was made up and 1 μg of the following activated 3′-adapter added: 5′-AppCTGTAGGCACCATCAAT–NH2-3′ (NEB 1315).

    Article Title: A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage
    Article Snippet: .. T4 RNA ligase catalyzes the ligation of an oligonucleotide bearing a 5′ phosphate group (the donor) to a second oligonucleotide bearing a free 3′-OH group (the acceptor). ..

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

    Article Title: Blocking of targeted microRNAs from next-generation sequencing libraries
    Article Snippet: This decrease in yield in the 3′ approach is likely due to the leftover ATP from the initial blocking ligation with T4 DNA Ligase inhibiting the truncated T4 RNA Ligase 2 in the subsequent ligation of the adaptor to the 3′ ends of the small RNA pool. .. Although truncated T4 RNA Ligase 2 cannot turnover ATP, ATP can still bind to the remnants of the active site, leading to inhibition of the enzyme (personal communication with NEB).

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage
    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. .. A typical large-scale ligation reaction using T4 DNA ligase was 10 μM in RNA fragments, 15 μM in DNA splint oligo, 10% PEG-4000 in 40 mM Tris–HCl pH = 7.8, 0.5 mM ATP, 10 mM MgCl2 , 10 mM DTT using 50 U T4 DNA ligase (fermentas) per nmol of RNA to be ligated or 2 μM final concentration of in-house produced T4 DNA ligase.

    Northern Blot:

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast
    Article Snippet: Quantification of northern blots was performed using a Storm 860 PhosohorImager and ImageQuant software (Molecular Dynamics). .. DNA ‘adaptor’ oligonucleotide (W242) carrying aminolinker at the 3′-end was ligated with the 3′-end of total RNA using 20U of T4 RNA ligase (NEB).

    Article Title: Human tRNA-derived small RNAs in the global regulation of RNA silencing
    Article Snippet: Fifteen-microliter reactions were incubated with the indicated enzymes at 37°C (Terminator Exonuclease: 30°C) for 60 min, acid phenol/chloroform extracted, ethanol precipitated, and resuspended for the second round of enzyme treatments, which was again followed by acid phenol/chloroform extraction, ethanol precipitation, and resuspension in PAGE loading buffer for Northern blot. .. Amounts of enzymes used: 15 units (U) of T4 PNK, 3′ phophatase ± (NEB M0201/m0236); 8 U of Tobacco Acid Pyrophosphatase (Epicentre Biotechnologies); 3 U of Terminator Exonuclease (Epicentre Biotechnologies); 4 U polyA polymerase (PAP; Ambion); and 15 U T4 RNA ligase (NEB).

    Inhibition:

    Article Title: Blocking of targeted microRNAs from next-generation sequencing libraries
    Article Snippet: .. Although truncated T4 RNA Ligase 2 cannot turnover ATP, ATP can still bind to the remnants of the active site, leading to inhibition of the enzyme (personal communication with NEB). .. Thus, a 3′ approach could likely be implemented without unwanted consequences if the reaction components of the blocking ligation were removed via column purification or some other suitable method.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast
    Article Snippet: DNA ‘adaptor’ oligonucleotide (W242) carrying aminolinker at the 3′-end was ligated with the 3′-end of total RNA using 20U of T4 RNA ligase (NEB). .. RNA was extracted with phenol: chloroform: isoamyl alcohol (v/v 25:24:1), precipitated and used as a template for cDNA synthesis using W243 primer complementary to the anchor sequence and the Enhanced Avian HS RT-PCR Kit (Sigma) according to manufacturer's instructions. cDNA was amplified using primers W241 and W243, the resulting PCR product was gel purified, cloned into pGEM-T Easy vector and sequenced using primer W241.

    Molecular Cloning:

    Article Title: Cloning and characterization of the extreme 5?-terminal sequences of the RNA genomes of GB virus C/hepatitis G virus
    Article Snippet: To avoid cross-contamination during manipulation, molecular cloning of the extreme 5′ noncoding region from the serum samples of three patients was done separately. .. 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).

    Isolation:

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast
    Article Snippet: The 3′ RACE assay was carried out on total RNA (20 μg) isolated from untreated cells and treated with 1mM H2 O2 . .. DNA ‘adaptor’ oligonucleotide (W242) carrying aminolinker at the 3′-end was ligated with the 3′-end of total RNA using 20U of T4 RNA ligase (NEB).

    Purification:

    Article Title: A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage
    Article Snippet: The phosphorylated dinucleotide ( ) was purified on a C18 reversed-phase high pressure liquid chromatography column (Vydac 201SP54) (0–30% acetonitrile/100–70% 0.1 M triethylammonium acetate, pH 7.0, over 30 min) and evaporated to dryness before further use. .. The 5-nt RNA 5′-CUCUU-3′ (40 nmol) and phosphorylated dinucleotide 24 (pC2′-X G5′-Y ) (20 nmol) were incubated together with 200 U of T4 RNA ligase (NEB), 10 µl DMSO, and 1× T4 RNA ligase buffer (50 mM Tris–HCl, pH 7.78, 10 mM MgCl2 , 10 mM dithiothreitol, 1 mM ATP) in a 100 μl reaction volume at 37°C for 6 h. The reaction mixture was worked up by PCI and ether extraction of the aqueous layers.

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast
    Article Snippet: DNA ‘adaptor’ oligonucleotide (W242) carrying aminolinker at the 3′-end was ligated with the 3′-end of total RNA using 20U of T4 RNA ligase (NEB). .. RNA was extracted with phenol: chloroform: isoamyl alcohol (v/v 25:24:1), precipitated and used as a template for cDNA synthesis using W243 primer complementary to the anchor sequence and the Enhanced Avian HS RT-PCR Kit (Sigma) according to manufacturer's instructions. cDNA was amplified using primers W241 and W243, the resulting PCR product was gel purified, cloned into pGEM-T Easy vector and sequenced using primer W241.

    Article Title: Blocking of targeted microRNAs from next-generation sequencing libraries
    Article Snippet: Although truncated T4 RNA Ligase 2 cannot turnover ATP, ATP can still bind to the remnants of the active site, leading to inhibition of the enzyme (personal communication with NEB). .. Thus, a 3′ approach could likely be implemented without unwanted consequences if the reaction components of the blocking ligation were removed via column purification or some other suitable method.

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage
    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. .. The reactions were subjected to HPLC purification followed by n -butanol extraction and lyophilization.

    Sequencing:

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast
    Article Snippet: Dideoxy-DNA sequencing was performed on PCR-templates prepared from genomic yeast DNA using the same primers as for primer extension (W235 and W237) and a fMol Seq kit (Promega) according to manufacturer's instructions. .. DNA ‘adaptor’ oligonucleotide (W242) carrying aminolinker at the 3′-end was ligated with the 3′-end of total RNA using 20U of T4 RNA ligase (NEB).

    Article Title: Uridylation by TUT4/7 Restricts Retrotransposition of Human LINE-1s
    Article Snippet: RNA (∼2-3 μg) for each sample was used in a ligation reaction containing 125 pmol RA3_15N 3′ adaptor (5′ preadenylated and individually barcoded with a 15N sequence). .. 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.

    Article Title: Blocking of targeted microRNAs from next-generation sequencing libraries
    Article Snippet: Paragraph title: Blocking hsa-miR-16–5p in sequencing libraries ... Although truncated T4 RNA Ligase 2 cannot turnover ATP, ATP can still bind to the remnants of the active site, leading to inhibition of the enzyme (personal communication with NEB).

    Polyacrylamide Gel Electrophoresis:

    Article Title: Uridylation by TUT4/7 Restricts Retrotransposition of Human LINE-1s
    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. .. To assess ligation efficiency, 1/10 of the sample was loaded onto 10% denaturing PAGE to separate free and RA3_15N-ligated RNA44.

    Article Title: Human tRNA-derived small RNAs in the global regulation of RNA silencing
    Article Snippet: Fifteen-microliter reactions were incubated with the indicated enzymes at 37°C (Terminator Exonuclease: 30°C) for 60 min, acid phenol/chloroform extracted, ethanol precipitated, and resuspended for the second round of enzyme treatments, which was again followed by acid phenol/chloroform extraction, ethanol precipitation, and resuspension in PAGE loading buffer for Northern blot. .. Amounts of enzymes used: 15 units (U) of T4 PNK, 3′ phophatase ± (NEB M0201/m0236); 8 U of Tobacco Acid Pyrophosphatase (Epicentre Biotechnologies); 3 U of Terminator Exonuclease (Epicentre Biotechnologies); 4 U polyA polymerase (PAP; Ambion); and 15 U T4 RNA ligase (NEB).

    Nested PCR:

    Article Title: Cloning and characterization of the extreme 5?-terminal sequences of the RNA genomes of GB virus C/hepatitis G virus
    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). .. A second-round nested PCR was done with another adapter primer (AP-2) complementary to the 5′ region of the adapter and the inner viral specific primer (VS-2).

    Plasmid Preparation:

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast
    Article Snippet: DNA ‘adaptor’ oligonucleotide (W242) carrying aminolinker at the 3′-end was ligated with the 3′-end of total RNA using 20U of T4 RNA ligase (NEB). .. RNA was extracted with phenol: chloroform: isoamyl alcohol (v/v 25:24:1), precipitated and used as a template for cDNA synthesis using W243 primer complementary to the anchor sequence and the Enhanced Avian HS RT-PCR Kit (Sigma) according to manufacturer's instructions. cDNA was amplified using primers W241 and W243, the resulting PCR product was gel purified, cloned into pGEM-T Easy vector and sequenced using primer W241.

    Software:

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast
    Article Snippet: Quantification of northern blots was performed using a Storm 860 PhosohorImager and ImageQuant software (Molecular Dynamics). .. DNA ‘adaptor’ oligonucleotide (W242) carrying aminolinker at the 3′-end was ligated with the 3′-end of total RNA using 20U of T4 RNA ligase (NEB).

    RNA Extraction:

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast
    Article Snippet: Paragraph title: RNA extraction and analysis ... DNA ‘adaptor’ oligonucleotide (W242) carrying aminolinker at the 3′-end was ligated with the 3′-end of total RNA using 20U of T4 RNA ligase (NEB).

    Ethanol Precipitation:

    Article Title: Human tRNA-derived small RNAs in the global regulation of RNA silencing
    Article Snippet: Fifteen-microliter reactions were incubated with the indicated enzymes at 37°C (Terminator Exonuclease: 30°C) for 60 min, acid phenol/chloroform extracted, ethanol precipitated, and resuspended for the second round of enzyme treatments, which was again followed by acid phenol/chloroform extraction, ethanol precipitation, and resuspension in PAGE loading buffer for Northern blot. .. Amounts of enzymes used: 15 units (U) of T4 PNK, 3′ phophatase ± (NEB M0201/m0236); 8 U of Tobacco Acid Pyrophosphatase (Epicentre Biotechnologies); 3 U of Terminator Exonuclease (Epicentre Biotechnologies); 4 U polyA polymerase (PAP; Ambion); and 15 U T4 RNA ligase (NEB).

    Produced:

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage
    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. .. A typical large-scale ligation reaction using T4 DNA ligase was 10 μM in RNA fragments, 15 μM in DNA splint oligo, 10% PEG-4000 in 40 mM Tris–HCl pH = 7.8, 0.5 mM ATP, 10 mM MgCl2 , 10 mM DTT using 50 U T4 DNA ligase (fermentas) per nmol of RNA to be ligated or 2 μM final concentration of in-house produced T4 DNA ligase.

    Concentration Assay:

    Article Title: Uridylation by TUT4/7 Restricts Retrotransposition of Human LINE-1s
    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. .. To assess ligation efficiency, 1/10 of the sample was loaded onto 10% denaturing PAGE to separate free and RA3_15N-ligated RNA44.

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage
    Article Snippet: RNA ligation using T4 RNA and DNA ligase Non-splinted T4 RNA based ligations were first performed on small scale reactions (typically 400 pmol RNA fragments in 10 μl reaction volume) mainly by optimizing the T4 RNA enzyme concentration (NEB) and testing the addition of BSA, whereas splinted T4 DNA based small scale ligation reactions (typically 200 pmol RNA fragments in 20 μl reaction volume) were performed by optimizing the T4 DNA enzyme concentration (NEB, fermentas or in-house ), the reaction time, the reaction temperature and testing the influence of PEG-4000. .. 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.

    other:

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis
    Article Snippet: Based on the fact that miRNA-mediated mRNA cleavage will generate 5′-monophosphate ends on the 3′ end cleavage product of the target mRNAs, it is possible to ligate RNA oligonucleotide adapter to the 5′ terminus of the 3′ end cleavage product by using T4 RNA ligase, whereas such RNA oligonucleotide adapter would not be ligated to mRNAs with conventional 5′ cap .

    Article Title: Highly specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification †Electronic supplementary information (ESI) available: Additional experimental materials, methods, DNA sequences and su
    Article Snippet: T4 RNA ligase 2, Splint R and HiScribe™ T7 High Yield RNA Synthesis Kit were purchased from New England Biolabs (Beijing, China).

    Article Title: Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation
    Article Snippet: The truncation derivatives tested include T4 RNA Ligase 2, truncated (T42t); T4 RNA Ligase 2, truncated K227Q; and T4 RNA Ligase 2, truncated KQ [ ].

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

    Article Title: Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA
    Article Snippet: T4 RNA ligase is reported to require a single unpaired nucleotide at the 3′-terminus.

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    New England Biolabs rna ligase
    <t>RT–PCR</t> and cRT–PCR detection of lariat (and/or circularized) versus linear wheat mitochondrial intron molecules. ( A ) RT–PCR across the excised intron junction using 24 h wheat mitochondrial <t>RNA</t> as template, either pre-treated with ligase (+L; lanes 2, 5, 7 and 9) or untreated (−L; lanes 1, 4, 6 and 8) generated products of sizes 450, 520, 630 and 400 bp (denoted by arrowheads) for rps7 mRNA, nad2 intron 4, cox2 intron and nad1 intron 2, respectively. Size markers are shown in lane 3. ( B ) Southern blot of gels (shown in A, lanes 4–9) using intron-specific oligomer probes for nad2 intron 4, cox2 intron and nad1 intron 2. ( C ) Schematic showing positions of primers (arrows 1 and 2) used for RT–PCR and cRT–PCR in panels (A) and (B). Branchpoint is shown by A. Dotted lines indicate regions of amplification. ( D–F ) Direct sequencing of RT–PCR products for (D) cox2 intron (+L), (E) nad1 intron 2 (−L) and (F) nad1 intron 2 (+L) using oligomers 6, 11 and 16, respectively (Supplementary data). Note that an inverted orientation of the sequencing gel is shown for (E). Arrows show positions of the 5′ terminal nucleotide of the introns, stars highlight short non-encoded A-rich stretches and the discrete non-encoded insert sequence is boxed in (E).
    Rna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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: 95/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    t4 rna ligase 2 - by Bioz Stars, 2020-02
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    Image Search Results


    RT–PCR and cRT–PCR detection of lariat (and/or circularized) versus linear wheat mitochondrial intron molecules. ( A ) RT–PCR across the excised intron junction using 24 h wheat mitochondrial RNA as template, either pre-treated with ligase (+L; lanes 2, 5, 7 and 9) or untreated (−L; lanes 1, 4, 6 and 8) generated products of sizes 450, 520, 630 and 400 bp (denoted by arrowheads) for rps7 mRNA, nad2 intron 4, cox2 intron and nad1 intron 2, respectively. Size markers are shown in lane 3. ( B ) Southern blot of gels (shown in A, lanes 4–9) using intron-specific oligomer probes for nad2 intron 4, cox2 intron and nad1 intron 2. ( C ) Schematic showing positions of primers (arrows 1 and 2) used for RT–PCR and cRT–PCR in panels (A) and (B). Branchpoint is shown by A. Dotted lines indicate regions of amplification. ( D–F ) Direct sequencing of RT–PCR products for (D) cox2 intron (+L), (E) nad1 intron 2 (−L) and (F) nad1 intron 2 (+L) using oligomers 6, 11 and 16, respectively (Supplementary data). Note that an inverted orientation of the sequencing gel is shown for (E). Arrows show positions of the 5′ terminal nucleotide of the introns, stars highlight short non-encoded A-rich stretches and the discrete non-encoded insert sequence is boxed in (E).

    Journal: Nucleic Acids Research

    Article Title: Multiple physical forms of excised group II intron RNAs in wheat mitochondria

    doi: 10.1093/nar/gkl328

    Figure Lengend Snippet: RT–PCR and cRT–PCR detection of lariat (and/or circularized) versus linear wheat mitochondrial intron molecules. ( A ) RT–PCR across the excised intron junction using 24 h wheat mitochondrial RNA as template, either pre-treated with ligase (+L; lanes 2, 5, 7 and 9) or untreated (−L; lanes 1, 4, 6 and 8) generated products of sizes 450, 520, 630 and 400 bp (denoted by arrowheads) for rps7 mRNA, nad2 intron 4, cox2 intron and nad1 intron 2, respectively. Size markers are shown in lane 3. ( B ) Southern blot of gels (shown in A, lanes 4–9) using intron-specific oligomer probes for nad2 intron 4, cox2 intron and nad1 intron 2. ( C ) Schematic showing positions of primers (arrows 1 and 2) used for RT–PCR and cRT–PCR in panels (A) and (B). Branchpoint is shown by A. Dotted lines indicate regions of amplification. ( D–F ) Direct sequencing of RT–PCR products for (D) cox2 intron (+L), (E) nad1 intron 2 (−L) and (F) nad1 intron 2 (+L) using oligomers 6, 11 and 16, respectively (Supplementary data). Note that an inverted orientation of the sequencing gel is shown for (E). Arrows show positions of the 5′ terminal nucleotide of the introns, stars highlight short non-encoded A-rich stretches and the discrete non-encoded insert sequence is boxed in (E).

    Article Snippet: As expected, the rps7 mRNA template resulted in an RT–PCR product of 450 bp only when pre-treated with RNA ligase ( , lane 1 versus lane 2, arrowhead).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Generated, Southern Blot, Amplification, Sequencing

    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