Structured Review

GE Healthcare t4 rna ligase
Effect of various modifications on the 3′ terminal nucleotide of a small RNA on <t>T4</t> RNA ligase- and yeast PAP-catalyzed reactions. ( a ) T4 RNA ligase-mediated ligation of various miR173 forms to an RNA linker. ( b ) Activity of yeast PAP on various forms of miR173 in the presence of 2 pmol [α- 32 P]-ATP. The ladders or smears represent products of PAP-catalyzed reaction. ( c ) Activity of yeast PAP on various forms of miR173 in the presence of 10 pmol [α- 32 P]-ATP.
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Images

1) Product Images from "HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2? OH of the 3? terminal nucleotide"

Article Title: HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2? OH of the 3? terminal nucleotide

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkj474

Effect of various modifications on the 3′ terminal nucleotide of a small RNA on T4 RNA ligase- and yeast PAP-catalyzed reactions. ( a ) T4 RNA ligase-mediated ligation of various miR173 forms to an RNA linker. ( b ) Activity of yeast PAP on various forms of miR173 in the presence of 2 pmol [α- 32 P]-ATP. The ladders or smears represent products of PAP-catalyzed reaction. ( c ) Activity of yeast PAP on various forms of miR173 in the presence of 10 pmol [α- 32 P]-ATP.
Figure Legend Snippet: Effect of various modifications on the 3′ terminal nucleotide of a small RNA on T4 RNA ligase- and yeast PAP-catalyzed reactions. ( a ) T4 RNA ligase-mediated ligation of various miR173 forms to an RNA linker. ( b ) Activity of yeast PAP on various forms of miR173 in the presence of 2 pmol [α- 32 P]-ATP. The ladders or smears represent products of PAP-catalyzed reaction. ( c ) Activity of yeast PAP on various forms of miR173 in the presence of 10 pmol [α- 32 P]-ATP.

Techniques Used: Ligation, Activity Assay

2) Product Images from "Combinatorial minimization and secondary structure determination of a nucleotide synthase ribozyme"

Article Title: Combinatorial minimization and secondary structure determination of a nucleotide synthase ribozyme

Journal: RNA

doi: 10.1261/rna.5500603

Selection scheme designed to retain sequence variation all the way to the 3′ end of RNA transcripts. ( A ) Pool RNA tethered to pRpp was incubated with 4S Ura. Active sequences react to form tethered 4-thiouridine. ( B ) Active sequences were purified away from unreactive sequences, using an APM gel. ( C ) Using T4 RNA ligase, purified ribozymes were then ligated to an adenylated DNA oligo containing an Ear I restriction site and 3′ RT-PCR primer binding region. ( D ) RT-PCR was performed to amplify ribozyme DNA sequence and add a T7 promoter (T7) to the 5′ end of the sequences. ( E ) Digestion with Ear I-generated DNA sequence lacking a fixed 3′ primer-binding site, which was then transcribed to generate RNA for another round of selection.
Figure Legend Snippet: Selection scheme designed to retain sequence variation all the way to the 3′ end of RNA transcripts. ( A ) Pool RNA tethered to pRpp was incubated with 4S Ura. Active sequences react to form tethered 4-thiouridine. ( B ) Active sequences were purified away from unreactive sequences, using an APM gel. ( C ) Using T4 RNA ligase, purified ribozymes were then ligated to an adenylated DNA oligo containing an Ear I restriction site and 3′ RT-PCR primer binding region. ( D ) RT-PCR was performed to amplify ribozyme DNA sequence and add a T7 promoter (T7) to the 5′ end of the sequences. ( E ) Digestion with Ear I-generated DNA sequence lacking a fixed 3′ primer-binding site, which was then transcribed to generate RNA for another round of selection.

Techniques Used: Selection, Sequencing, Incubation, Purification, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Generated

3) Product Images from "RISC is a 5? phosphomonoester-producing RNA endonuclease"

Article Title: RISC is a 5? phosphomonoester-producing RNA endonuclease

Journal: Genes & Development

doi: 10.1101/gad.1187904

Target RNA is cleaved endonucleolytically producing 5′-phosphate and 3′-hydroxyl termini. ( A ) Preparation of site-specifically labeled substrates and cleavage assay. 5′- 32 P-labeled and 3′ aminolinker (L) protected 12-nt oligoribonucleotide was ligated to nonphosphorylated 9-nt oligoribonucleotide using T4 RNA ligase. An aliquot of the ligation product was further 5′- 32 P-labeled using T4 polynucleotide kinase. The purified substrates were incubated with affinity-purified RISC programmed with single-stranded guide siRNA. ( B ) PhosphorImaging of cleavage reactions incubated for 2 h at 30°C, and resolved on a 20% denaturing polyacrylamide gel. 5′- 32 P-labeled 9- and 12-nt oligoribonucleotides were loaded as marker in lanes 1 and 2 , respectively. The cleavage reactions with single- and double-labeled 21-nt substrate are loaded in lanes 4 and 5 , respectively. Lane 3 contains the 12-nt cleavage product isolated from a prior cleavage reaction. ( C ) Two-dimensional thin layer chromatography analysis of the ribonuclease T2-digested RISC-cleavage product. The oval depicts the unlabeled pAp as detected by UV shadowing. The radioactive signal comigrates with the 5′ 32 pAp released by ribonuclease T2 digestion from the gel-purified 12-nt cleavage product.
Figure Legend Snippet: Target RNA is cleaved endonucleolytically producing 5′-phosphate and 3′-hydroxyl termini. ( A ) Preparation of site-specifically labeled substrates and cleavage assay. 5′- 32 P-labeled and 3′ aminolinker (L) protected 12-nt oligoribonucleotide was ligated to nonphosphorylated 9-nt oligoribonucleotide using T4 RNA ligase. An aliquot of the ligation product was further 5′- 32 P-labeled using T4 polynucleotide kinase. The purified substrates were incubated with affinity-purified RISC programmed with single-stranded guide siRNA. ( B ) PhosphorImaging of cleavage reactions incubated for 2 h at 30°C, and resolved on a 20% denaturing polyacrylamide gel. 5′- 32 P-labeled 9- and 12-nt oligoribonucleotides were loaded as marker in lanes 1 and 2 , respectively. The cleavage reactions with single- and double-labeled 21-nt substrate are loaded in lanes 4 and 5 , respectively. Lane 3 contains the 12-nt cleavage product isolated from a prior cleavage reaction. ( C ) Two-dimensional thin layer chromatography analysis of the ribonuclease T2-digested RISC-cleavage product. The oval depicts the unlabeled pAp as detected by UV shadowing. The radioactive signal comigrates with the 5′ 32 pAp released by ribonuclease T2 digestion from the gel-purified 12-nt cleavage product.

Techniques Used: Labeling, Cleavage Assay, Ligation, Purification, Incubation, Affinity Purification, Marker, Isolation, Thin Layer Chromatography

4) Product Images from "RNA-Dependent DNA Binding Activity of the Pur Factor, Potentially Involved in DNA Replication and Gene Transcription"

Article Title: RNA-Dependent DNA Binding Activity of the Pur Factor, Potentially Involved in DNA Replication and Gene Transcription

Journal: Gene Expression

doi:

RNAs interacting with Pur are present in rabbit retic-ulocyte lysates. (A) Sensitivity to RNase A of the PUR binding activity of the in vitro translated Purα protein. The ability of an in vitro-translated Purα protein to bind 32 P-labeled PUR oligonucleotides was examined by mobility shift assay. Binding reactions were done with the reticulocyte lysate immediately after the translation reaction (lane 1), or after an additional 30-min incubation at 37°C in the absence (lane 2) or presence (lane 3) of RNase A. (B) Presence in the rabbit reticulocyte lysate of RNAs similar to the Pur-associated RNAs of fraction 14 of RSV-infected QEF nuclear extracts. Total RNAs were prepared from fraction 14 of nuclear extracts from RSV-infected QEF (lane 1), or the rabbit reticulocyte lysate (lane 2), and 3′ end labeled using the T4 RNA polymerase, and run in parallel in a denaturing 20% polyacrylamide gel, along with 22, 27, and 32 mer radioactive oligonucleotides as molecular weight markers. Identical 28- and 29-bases-long RNAs, similar to those previously found associated with the PUR complexes, are present in both preparations, and are indicated by arrows.
Figure Legend Snippet: RNAs interacting with Pur are present in rabbit retic-ulocyte lysates. (A) Sensitivity to RNase A of the PUR binding activity of the in vitro translated Purα protein. The ability of an in vitro-translated Purα protein to bind 32 P-labeled PUR oligonucleotides was examined by mobility shift assay. Binding reactions were done with the reticulocyte lysate immediately after the translation reaction (lane 1), or after an additional 30-min incubation at 37°C in the absence (lane 2) or presence (lane 3) of RNase A. (B) Presence in the rabbit reticulocyte lysate of RNAs similar to the Pur-associated RNAs of fraction 14 of RSV-infected QEF nuclear extracts. Total RNAs were prepared from fraction 14 of nuclear extracts from RSV-infected QEF (lane 1), or the rabbit reticulocyte lysate (lane 2), and 3′ end labeled using the T4 RNA polymerase, and run in parallel in a denaturing 20% polyacrylamide gel, along with 22, 27, and 32 mer radioactive oligonucleotides as molecular weight markers. Identical 28- and 29-bases-long RNAs, similar to those previously found associated with the PUR complexes, are present in both preparations, and are indicated by arrows.

Techniques Used: Binding Assay, Activity Assay, In Vitro, Labeling, Mobility Shift, Incubation, Infection, Molecular Weight

5) Product Images from "Physical and Functional Interactions of the Arf Tumor Suppressor Protein with Nucleophosmin/B23"

Article Title: Physical and Functional Interactions of the Arf Tumor Suppressor Protein with Nucleophosmin/B23

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.3.985-996.2004

Arf forms complexes with NPM. (A, upper two panels) Lysates prepared from NIH 3T3 and MT-Arf cells treated with zinc sulfate for 24 h (+) or not (−) were precipitated with antibodies to NPM (lanes 5 to 8), p19 Arf (lanes 9 to 12), or control IgG (lanes 13 and 14). Denatured immune complexes electrophoretically separated on gels were transferred to a membrane and blotted with the same antibodies. Cell lysates (10%) separated on denaturing gels were directly blotted with antibodies to NPM and p19 Arf in order to estimate levels of the expressed proteins (lanes 1 to 4; see text). (A, lower panel) RNAs eluted from immune complexes were labeled with [ 32 P]pCp and T4 RNA ligase, separated on a denaturing gel, and detected by autoradiography. (B) Primary MEFs established from day 15 embryos were propagated on a 3T3 protocol for seven passages. Cell lysates expressing both p19 Arf and NPM (lane 1) were precipitated with control antibodies (lane 2) or with antibodies to NPM or Arf (lanes 3 and 4). Proteins separated on denaturing gels were immunoblotted with the same antibodies, indicated at the right of the panel. (C) Arf/Mdm2/p53 triple knockout (TKO) MEFs infected with retroviruses encoding p19 Arf or the p19 Arf Δ2-14 mutant were lysed and precipitated with antibodies to p19 Arf (lanes 4 to 6) or nonimmune rabbit serum (NRS) (lanes 7 to 9) and subjected to immunoblotting with antibodies to Arf (top panel) or NPM (bottom panel). A portion of the cell lysate was used to demonstrate levels of protein expression (lanes 1 to 3) as in panel A.
Figure Legend Snippet: Arf forms complexes with NPM. (A, upper two panels) Lysates prepared from NIH 3T3 and MT-Arf cells treated with zinc sulfate for 24 h (+) or not (−) were precipitated with antibodies to NPM (lanes 5 to 8), p19 Arf (lanes 9 to 12), or control IgG (lanes 13 and 14). Denatured immune complexes electrophoretically separated on gels were transferred to a membrane and blotted with the same antibodies. Cell lysates (10%) separated on denaturing gels were directly blotted with antibodies to NPM and p19 Arf in order to estimate levels of the expressed proteins (lanes 1 to 4; see text). (A, lower panel) RNAs eluted from immune complexes were labeled with [ 32 P]pCp and T4 RNA ligase, separated on a denaturing gel, and detected by autoradiography. (B) Primary MEFs established from day 15 embryos were propagated on a 3T3 protocol for seven passages. Cell lysates expressing both p19 Arf and NPM (lane 1) were precipitated with control antibodies (lane 2) or with antibodies to NPM or Arf (lanes 3 and 4). Proteins separated on denaturing gels were immunoblotted with the same antibodies, indicated at the right of the panel. (C) Arf/Mdm2/p53 triple knockout (TKO) MEFs infected with retroviruses encoding p19 Arf or the p19 Arf Δ2-14 mutant were lysed and precipitated with antibodies to p19 Arf (lanes 4 to 6) or nonimmune rabbit serum (NRS) (lanes 7 to 9) and subjected to immunoblotting with antibodies to Arf (top panel) or NPM (bottom panel). A portion of the cell lysate was used to demonstrate levels of protein expression (lanes 1 to 3) as in panel A.

Techniques Used: Labeling, Autoradiography, Expressing, Triple Knockout, Infection, Mutagenesis

Related Articles

Clone Assay:

Article Title: Identification and characterization of new miRNAs cloned from normal mouse mammary gland
Article Snippet: Paragraph title: Small RNA isolation and cloning ... 3' adapter (5' phosphorylated) was ligated to the RNA fraction in presence of T4 RNA ligase (Amersham-Pharmacia).

Amplification:

Article Title: An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation
Article Snippet: First, the template was assembled by polymerase chain reaction using the 5′-half sense primer (5′-GCGTAATACGACTCACTATAGCGCGGATAGCTCAGTCG-3′) and the 5′-half antisense primer (5′-TCAATCCCCTGCTCTACCGACTGAGCTATCCG-3′); second, the product of this assembly was further amplified using the sense T7 promoter primer (5′-GCGTAATACGACTCACTATAG-3′) and a methylated reverse primer (5′-mUmCAATCCCCTGCTCTACCGAC-3′). .. To generate full-length 2AP-tRNA, 15 µM 5′-half tRNA was annealed in 1.5× excess to 2AP-3′-half tRNA in 1× T4 RNA ligase buffer (New England Biolabs) by heating at 95°C for 3 min, 65°C for 10 min and then slowly cooling from 65 to 20°C over 45 min. T4 RNA ligase (0.02 U/ml) was added to the mixture followed by incubation at 37°C for 4 h. Ligated tRNA was purified by urea-PAGE, ultraviolet-shadowing and electroelution (EluTrap electroelution system, Whatman).

Article Title: NtcA-Regulated Heterocyst Differentiation Genes hetC and devB from Anabaena sp. Strain PCC 7120 Exhibit a Similar Tandem Promoter Arrangement
Article Snippet: Reverse transcription-PCR amplification of transcripts ligated to short 5′ RNA adaptors was carried out as described previously ( ). .. Sixty picomoles of 5′ RNA adaptor (Table ) (Sigma Proligo) was added to the RNA sample, and ligation was performed in the presence of 80 units of T4 RNA ligase (Amersham) for 16 h at 15°C.

Article Title: Genomic Characterization of a Novel Virus of the Family Tymoviridae Isolated from Mosquitoes
Article Snippet: The 5′-RACE method was employed using a commercially available kit (Invitrogen) with the Universal Amplification Primer (UAP) and CuTLV segment-specific primer R1 , according to the manufacturer's specifications. .. Briefly, the 3′-end of the viral RNA was ligated with an adapter (5′-P-GTCGATCACGCGATCGAACGGTCGC TGAG-3′) using T4 RNA ligase (Amersham).

Article Title: Identification and characterization of new miRNAs cloned from normal mouse mammary gland
Article Snippet: 3' adapter (5' phosphorylated) was ligated to the RNA fraction in presence of T4 RNA ligase (Amersham-Pharmacia). .. The sample was again size fractionated using 15% PAGE before to be reverse transcribed using primer complementary to the 3' adapter sequence and PCR amplified using primers on both adapters.

Synthesized:

Article Title: HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2? OH of the 3? terminal nucleotide
Article Snippet: Ligation with T4 RNA ligase Synthesized miR173 RNA standard and miR173 with a methyl group on either the 2′ or 3′ OH (miR173-2′OMe or miR173-3′OMe) were treated with calf intestine alkaline phosphatase (New England Biolabs) to remove the 5′P to prevent self-ligation. .. Ligation of the miR173 RNAs to an RNA linker (5′-pUAUGAAGCC), in which the 3′ OH is blocked by a C-3 spacer (CH2 CH2 CH2 OH), was performed at 16°C for 16 h in a 10 µl mixture containing 20 U T4 RNA ligase (Amersham Pharmacia), 1 ng of dephosphorylated miR173 and 1 µg RNA linker.

Autoradiography:

Article Title: Facile FMR1 mRNA structure regulation by interruptions in CGG repeats
Article Snippet: .. To label the 3′ end, the RNA fragments were incubated with 20 U of T4 RNA ligase (Epicentre Technologies) and 30 μCi of [32 P]pCp (Amersham Corp.), at 4°C for 16–18 h. The labeled RNAs were purified by electrophoresis on denaturing 10% polyacrylamide gels, localized on the gels by autoradiography and recovered as described earlier ( ). .. Analysis of RNA conformation using native PAGE In order to check for structure homogeneity of the RNA molecules, all labeled transcripts were analyzed on 5–10% polyacrylamide gels (400/300/0.8 mm, acrylamide/bisakrylamide–29/1) in non-denaturing conditions.

Article Title: 2?-O-methylation stabilizes Piwi-associated small RNAs and ensures DNA elimination in Tetrahymena
Article Snippet: The purified scnRNAs were ligated to [5′-32 P]pCp using T4 RNA ligase (Amersham). .. The labeled dinucleotides were visualized by autoradiography, while the synthetic dinucleotide standards were visualized by UV shadowing.

Electrophoresis:

Article Title: RNA-Dependent DNA Binding Activity of the Pur Factor, Potentially Involved in DNA Replication and Gene Transcription
Article Snippet: .. After phenol extraction and ethanol precipitation in the presence of 1 μ g glycogen, RNAs were ligated to [32 P]Cp (100 μ Ci, Amersham) by the T4 RNA ligase (20 U) (Biolabs) in 50 mM Tris-Cl− (pH 7.8), 10 mM MgCl2 , 10 mM β -mercaptoethanol, 1 mM ATP, and 1 mM hexamine cobalt chloride, in the presence of 20 U RNasin (Promega) in a final volume of 30 μ l at 10°C for 16 h. Aliquots (10 μ l) of the ligation reactions were mixed to an equal volume of formamide buffer and incubated at 90 °C for 5 min prior to electrophoresis. .. The 1.1-kb-long Purα full-length cDNA present in the insert of the λgt11 clone λAB6 , kindly provided by Dr. Johnson, was subcloned into the EcoRI site of the pBSK plasmid.

Article Title: An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation
Article Snippet: Following ethanol precipitation, the RNA was purified using the BioRad Model 491 Prep Cell continuous electrophoresis purification system. .. To generate full-length 2AP-tRNA, 15 µM 5′-half tRNA was annealed in 1.5× excess to 2AP-3′-half tRNA in 1× T4 RNA ligase buffer (New England Biolabs) by heating at 95°C for 3 min, 65°C for 10 min and then slowly cooling from 65 to 20°C over 45 min. T4 RNA ligase (0.02 U/ml) was added to the mixture followed by incubation at 37°C for 4 h. Ligated tRNA was purified by urea-PAGE, ultraviolet-shadowing and electroelution (EluTrap electroelution system, Whatman).

Article Title: Facile FMR1 mRNA structure regulation by interruptions in CGG repeats
Article Snippet: .. To label the 3′ end, the RNA fragments were incubated with 20 U of T4 RNA ligase (Epicentre Technologies) and 30 μCi of [32 P]pCp (Amersham Corp.), at 4°C for 16–18 h. The labeled RNAs were purified by electrophoresis on denaturing 10% polyacrylamide gels, localized on the gels by autoradiography and recovered as described earlier ( ). .. Analysis of RNA conformation using native PAGE In order to check for structure homogeneity of the RNA molecules, all labeled transcripts were analyzed on 5–10% polyacrylamide gels (400/300/0.8 mm, acrylamide/bisakrylamide–29/1) in non-denaturing conditions.

Microarray:

Article Title: MicroRNAs Regulate Human Brain Endothelial Cell-Barrier Function in Inflammation: Implications for Multiple Sclerosis
Article Snippet: .. Total RNA (100 ng) was labeled with pCp-Cy3 using T4 RNA ligase (GE Healthcare) and the Agilent human microRNA microarray kit annotated in the Sanger miRbase (release 12.0) containing probes for 939 human microRNAs was performed according to the Agilent's microRNA microarray profiling. .. GeneSpring GX 9 software (Agilent Technologies) was used for value extraction.

Incubation:

Article Title: RNA-Dependent DNA Binding Activity of the Pur Factor, Potentially Involved in DNA Replication and Gene Transcription
Article Snippet: .. After phenol extraction and ethanol precipitation in the presence of 1 μ g glycogen, RNAs were ligated to [32 P]Cp (100 μ Ci, Amersham) by the T4 RNA ligase (20 U) (Biolabs) in 50 mM Tris-Cl− (pH 7.8), 10 mM MgCl2 , 10 mM β -mercaptoethanol, 1 mM ATP, and 1 mM hexamine cobalt chloride, in the presence of 20 U RNasin (Promega) in a final volume of 30 μ l at 10°C for 16 h. Aliquots (10 μ l) of the ligation reactions were mixed to an equal volume of formamide buffer and incubated at 90 °C for 5 min prior to electrophoresis. .. The 1.1-kb-long Purα full-length cDNA present in the insert of the λgt11 clone λAB6 , kindly provided by Dr. Johnson, was subcloned into the EcoRI site of the pBSK plasmid.

Article Title: Conserved features of Y RNAs: a comparison of experimentally derived secondary structures
Article Snippet: 3′-End labeling was achieved via ligating [32 P]pCp using T4 RNA ligase (Amersham Pharmacia Biotech) for 1 h at 37°C. .. After a 5 min incubation at 37°C, 0.1 mM of each (unlabeled) nucleotide was added and incubated for 1 h. Each of the labeled products was isolated from a 10% PAA/8 M urea gel.

Article Title: An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation
Article Snippet: .. To generate full-length 2AP-tRNA, 15 µM 5′-half tRNA was annealed in 1.5× excess to 2AP-3′-half tRNA in 1× T4 RNA ligase buffer (New England Biolabs) by heating at 95°C for 3 min, 65°C for 10 min and then slowly cooling from 65 to 20°C over 45 min. T4 RNA ligase (0.02 U/ml) was added to the mixture followed by incubation at 37°C for 4 h. Ligated tRNA was purified by urea-PAGE, ultraviolet-shadowing and electroelution (EluTrap electroelution system, Whatman). .. Fluorescence spectroscopy and stopped-flow experiments 2AP-tRNA (0.05–0.2 µM) diluted in 1× TAKEM4 buffer was excited at 325 nm, titrated with TruB and the emission was measured from 340 to 400 nm using a Quanta Master 60 fluorescence spectrometer (Photon Technology International).

Article Title: Facile FMR1 mRNA structure regulation by interruptions in CGG repeats
Article Snippet: .. To label the 3′ end, the RNA fragments were incubated with 20 U of T4 RNA ligase (Epicentre Technologies) and 30 μCi of [32 P]pCp (Amersham Corp.), at 4°C for 16–18 h. The labeled RNAs were purified by electrophoresis on denaturing 10% polyacrylamide gels, localized on the gels by autoradiography and recovered as described earlier ( ). .. Analysis of RNA conformation using native PAGE In order to check for structure homogeneity of the RNA molecules, all labeled transcripts were analyzed on 5–10% polyacrylamide gels (400/300/0.8 mm, acrylamide/bisakrylamide–29/1) in non-denaturing conditions.

Article Title: Physical and Functional Interactions of the Arf Tumor Suppressor Protein with Nucleophosmin/B23
Article Snippet: For analysis of coprecipitating RNAs, immune complexes were suspended and incubated for 30 min at 37°C in 7 mM Tris HCl, pH 7.5, containing 0.7 mM EDTA, 20 mM NaCl, 0.7% sodium dodecyl sulfate, and 30 μg of proteinase K per ml (Sigma Chemicals). .. RNAs were recovered by phenol-chloroform extraction followed by ethanol precipitation in 0.3 M sodium acetate containing 20 μg of glycogen (Roche, Basel, Switzerland), and end-labeled with 40 μCi of [32 P]cytidine 3′, 5′-bis(phosphate) ([32 ]pCp) (3,000 Ci/mmol; ICN, Costa Mesa, Calif.) with T4 RNA ligase (Amersham Biosciences) as described ( ).

Article Title: Combinatorial minimization and secondary structure determination of a nucleotide synthase ribozyme
Article Snippet: DNA was transcribed into RNA (with or without [32 P]-α-UTP, 40 mM Tris pH 8.6, 26 mM MgCl2 , 5 mM DTT, 2.5 mM Spermidine, 0.01% TritonX-100, 8 mM GTP, 4 mM ATP, 4 mM CTP, 2 mM UTP, and 5 U/μL T7 RNA polymerase, incubated for 1 h at 37°C), and the RNA was gel-purified on 5% polyacrylamide gels. .. RNA was precipitated and then ligated at the 3′ end to pRpp using adenylated pRpp (AppRpp) and T4 RNA ligase (Amersham Pharmacia).

Hybridization:

Article Title: HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2? OH of the 3? terminal nucleotide
Article Snippet: Ligation of the miR173 RNAs to an RNA linker (5′-pUAUGAAGCC), in which the 3′ OH is blocked by a C-3 spacer (CH2 CH2 CH2 OH), was performed at 16°C for 16 h in a 10 µl mixture containing 20 U T4 RNA ligase (Amersham Pharmacia), 1 ng of dephosphorylated miR173 and 1 µg RNA linker. .. Free miR173 and miR173 ligated to the RNA linker were resolved by gel electrophoresis and detected by RNA filter hybridization using a probe complementary to miR173.

Flow Cytometry:

Article Title: An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation
Article Snippet: The RNA (1.44 ml) was loaded on a 5-cm 12% urea-PAGE (28 mm diameter) and a constant voltage of 250 V was applied while eluting with 1× Tris/Borate/EDTA (TBE) buffer at a flow rate of 0.75 ml/min at the bottom of the gel. .. To generate full-length 2AP-tRNA, 15 µM 5′-half tRNA was annealed in 1.5× excess to 2AP-3′-half tRNA in 1× T4 RNA ligase buffer (New England Biolabs) by heating at 95°C for 3 min, 65°C for 10 min and then slowly cooling from 65 to 20°C over 45 min. T4 RNA ligase (0.02 U/ml) was added to the mixture followed by incubation at 37°C for 4 h. Ligated tRNA was purified by urea-PAGE, ultraviolet-shadowing and electroelution (EluTrap electroelution system, Whatman).

Ligation:

Article Title: RNA-Dependent DNA Binding Activity of the Pur Factor, Potentially Involved in DNA Replication and Gene Transcription
Article Snippet: .. After phenol extraction and ethanol precipitation in the presence of 1 μ g glycogen, RNAs were ligated to [32 P]Cp (100 μ Ci, Amersham) by the T4 RNA ligase (20 U) (Biolabs) in 50 mM Tris-Cl− (pH 7.8), 10 mM MgCl2 , 10 mM β -mercaptoethanol, 1 mM ATP, and 1 mM hexamine cobalt chloride, in the presence of 20 U RNasin (Promega) in a final volume of 30 μ l at 10°C for 16 h. Aliquots (10 μ l) of the ligation reactions were mixed to an equal volume of formamide buffer and incubated at 90 °C for 5 min prior to electrophoresis. .. The 1.1-kb-long Purα full-length cDNA present in the insert of the λgt11 clone λAB6 , kindly provided by Dr. Johnson, was subcloned into the EcoRI site of the pBSK plasmid.

Article Title: HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2? OH of the 3? terminal nucleotide
Article Snippet: .. Ligation of the miR173 RNAs to an RNA linker (5′-pUAUGAAGCC), in which the 3′ OH is blocked by a C-3 spacer (CH2 CH2 CH2 OH), was performed at 16°C for 16 h in a 10 µl mixture containing 20 U T4 RNA ligase (Amersham Pharmacia), 1 ng of dephosphorylated miR173 and 1 µg RNA linker. .. Free miR173 and miR173 ligated to the RNA linker were resolved by gel electrophoresis and detected by RNA filter hybridization using a probe complementary to miR173.

Article Title: NtcA-Regulated Heterocyst Differentiation Genes hetC and devB from Anabaena sp. Strain PCC 7120 Exhibit a Similar Tandem Promoter Arrangement
Article Snippet: .. Sixty picomoles of 5′ RNA adaptor (Table ) (Sigma Proligo) was added to the RNA sample, and ligation was performed in the presence of 80 units of T4 RNA ligase (Amersham) for 16 h at 15°C. ..

Article Title: RISC is a 5? phosphomonoester-producing RNA endonuclease
Article Snippet: Paragraph title: RNA ligation ... Site-specific internally labeled substrate RNA was prepared by incubating 2 pmol 5′-32 P-phosphorylated and linker-modified 12-nt RNA with 20 pmol 9-nt RNA, and 40 units of T4 RNA ligase (Amersham) in 10 mM MgCl2 /10 mM 2-mercaptoethanol/50 mM Tris-HCl (pH 7.6)/0.1 mg/mL acetylated bovine serum albumin (Ac-BS, Sigma)/0.2 mM ATP/15% DMSO in a final volume of 20 μL.

Article Title: Combinatorial minimization and secondary structure determination of a nucleotide synthase ribozyme
Article Snippet: RNA was precipitated and then ligated at the 3′ end to pRpp using adenylated pRpp (AppRpp) and T4 RNA ligase (Amersham Pharmacia). .. Ligation conditions were 50 mM HEPES pH 8.3, 10 mM MgCl2 , 3.3 mM dithiothreitol (DTT), 10 μg/mL BSA, 8.3% v/v glycerol, 65 μM AppRpp, 1 U/μL enzyme, for 4 h at 23°C.

Sedimentation:

Article Title: Physical and Functional Interactions of the Arf Tumor Suppressor Protein with Nucleophosmin/B23
Article Snippet: Cells were suspended in NET2 buffer (50 mM Tris HCl, pH 7.4, containing 150 mM NaCl and 0.05% Nonidet P-40) and sonicated three times for 30 s. Lysates cleared by sedimentation in a microcentrifuge at 14,000 rpm for 10 min were precipitated for 3 h at 4°C with anti-NPM (Zymed, South San Francisco, Calif.), M2 anti-Flag (Sigma Chemicals), or antibodies to p19Arf . .. RNAs were recovered by phenol-chloroform extraction followed by ethanol precipitation in 0.3 M sodium acetate containing 20 μg of glycogen (Roche, Basel, Switzerland), and end-labeled with 40 μCi of [32 P]cytidine 3′, 5′-bis(phosphate) ([32 ]pCp) (3,000 Ci/mmol; ICN, Costa Mesa, Calif.) with T4 RNA ligase (Amersham Biosciences) as described ( ).

Generated:

Article Title: Global analysis of the mammalian RNA degradome reveals widespread miRNA-dependent and miRNA-independent endonucleolytic cleavage
Article Snippet: Gene specific 5′-RACE For the identification of RISC, 0.3 µg of 5′-RACE adapter (Ambion First Choice RLM-RACE kit; Applied Biosystems) was ligated to 2 µg DNase I-treated total RNA from human rectal mucosa, overnight at 14°C, using T4 RNA Ligase (GE Life Sciences). .. Random-primed cDNA was generated from one-fifth of the ligated RNA using Superscript II (Invitrogen) according to the manufacturer’s instructions.

Random Primed:

Article Title: Global analysis of the mammalian RNA degradome reveals widespread miRNA-dependent and miRNA-independent endonucleolytic cleavage
Article Snippet: Gene specific 5′-RACE For the identification of RISC, 0.3 µg of 5′-RACE adapter (Ambion First Choice RLM-RACE kit; Applied Biosystems) was ligated to 2 µg DNase I-treated total RNA from human rectal mucosa, overnight at 14°C, using T4 RNA Ligase (GE Life Sciences). .. Random-primed cDNA was generated from one-fifth of the ligated RNA using Superscript II (Invitrogen) according to the manufacturer’s instructions.

Polymerase Chain Reaction:

Article Title: An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation
Article Snippet: First, the template was assembled by polymerase chain reaction using the 5′-half sense primer (5′-GCGTAATACGACTCACTATAGCGCGGATAGCTCAGTCG-3′) and the 5′-half antisense primer (5′-TCAATCCCCTGCTCTACCGACTGAGCTATCCG-3′); second, the product of this assembly was further amplified using the sense T7 promoter primer (5′-GCGTAATACGACTCACTATAG-3′) and a methylated reverse primer (5′-mUmCAATCCCCTGCTCTACCGAC-3′). .. To generate full-length 2AP-tRNA, 15 µM 5′-half tRNA was annealed in 1.5× excess to 2AP-3′-half tRNA in 1× T4 RNA ligase buffer (New England Biolabs) by heating at 95°C for 3 min, 65°C for 10 min and then slowly cooling from 65 to 20°C over 45 min. T4 RNA ligase (0.02 U/ml) was added to the mixture followed by incubation at 37°C for 4 h. Ligated tRNA was purified by urea-PAGE, ultraviolet-shadowing and electroelution (EluTrap electroelution system, Whatman).

Article Title: IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION
Article Snippet: A series of successive 10 nucleotide deletions were made to the clone 4–15 RNA template using appropriately designed PCR primers. .. Alternatively, RNAs were 3′ end labeled using T4 RNA ligase and [32 P]pCp (GE Healthcare) using the protocol supplied by the enzyme manufacturer (Ambion).

Article Title: Global analysis of the mammalian RNA degradome reveals widespread miRNA-dependent and miRNA-independent endonucleolytic cleavage
Article Snippet: Gene specific 5′-RACE For the identification of RISC, 0.3 µg of 5′-RACE adapter (Ambion First Choice RLM-RACE kit; Applied Biosystems) was ligated to 2 µg DNase I-treated total RNA from human rectal mucosa, overnight at 14°C, using T4 RNA Ligase (GE Life Sciences). .. The cDNA was then used as template in a PCR reaction containing 0.2 µM 5′-RACE Outer Primer (FirstChoice® RLM-RACE kit; Applied Biosystems; 5′-GCTGATGGCGATGAATGAACACTG-3′) and 0.2 µM RICS-specific primer (5′-AACAGTCCACTGTCCAGCAGAGG-3′).

Article Title: Genomic Characterization of a Novel Virus of the Family Tymoviridae Isolated from Mosquitoes
Article Snippet: Briefly, the 3′-end of the viral RNA was ligated with an adapter (5′-P-GTCGATCACGCGATCGAACGGTCGC TGAG-3′) using T4 RNA ligase (Amersham). .. PCR was performed with primer F8 ( ) and the short primer under the following conditions: denaturation at 94°C for 5 min; 30 cycles of amplification (94°C for 30 s, 58°C for 30 s, 72°C for 2 min); and a final extension for 10 min at 72°C.

Article Title: Identification and characterization of new miRNAs cloned from normal mouse mammary gland
Article Snippet: [ ] without the step of concatemerization of the PCR products. .. 3' adapter (5' phosphorylated) was ligated to the RNA fraction in presence of T4 RNA ligase (Amersham-Pharmacia).

Article Title: Combinatorial minimization and secondary structure determination of a nucleotide synthase ribozyme
Article Snippet: PCR reactions (30 mL, 10 mM Tris pH 8.3, 50 mM KCl, 4 mM MgCl2 , 0.2 mM dNTPs, 0.01% gelatin, Taq enzyme, and 1 μM primers) were performed using the following primers: a1-5′-ttctaatacgactcactataggataatgcgcacaagt and 5′-gagcgtggacag gtgcc; a2-5′-ctatcccgactggcacc and 5′-tcgtcgtaggctcttca. .. RNA was precipitated and then ligated at the 3′ end to pRpp using adenylated pRpp (AppRpp) and T4 RNA ligase (Amersham Pharmacia).

Sonication:

Article Title: Physical and Functional Interactions of the Arf Tumor Suppressor Protein with Nucleophosmin/B23
Article Snippet: Cells were suspended in NET2 buffer (50 mM Tris HCl, pH 7.4, containing 150 mM NaCl and 0.05% Nonidet P-40) and sonicated three times for 30 s. Lysates cleared by sedimentation in a microcentrifuge at 14,000 rpm for 10 min were precipitated for 3 h at 4°C with anti-NPM (Zymed, South San Francisco, Calif.), M2 anti-Flag (Sigma Chemicals), or antibodies to p19Arf . .. RNAs were recovered by phenol-chloroform extraction followed by ethanol precipitation in 0.3 M sodium acetate containing 20 μg of glycogen (Roche, Basel, Switzerland), and end-labeled with 40 μCi of [32 P]cytidine 3′, 5′-bis(phosphate) ([32 ]pCp) (3,000 Ci/mmol; ICN, Costa Mesa, Calif.) with T4 RNA ligase (Amersham Biosciences) as described ( ).

Paper Chromatography:

Article Title: IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION
Article Snippet: The radiolabeled RNAs were analyzed using paper chromatography and cellulose-packed column assays as described above. .. Alternatively, RNAs were 3′ end labeled using T4 RNA ligase and [32 P]pCp (GE Healthcare) using the protocol supplied by the enzyme manufacturer (Ambion).

Nucleic Acid Electrophoresis:

Article Title: HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2? OH of the 3? terminal nucleotide
Article Snippet: Ligation of the miR173 RNAs to an RNA linker (5′-pUAUGAAGCC), in which the 3′ OH is blocked by a C-3 spacer (CH2 CH2 CH2 OH), was performed at 16°C for 16 h in a 10 µl mixture containing 20 U T4 RNA ligase (Amersham Pharmacia), 1 ng of dephosphorylated miR173 and 1 µg RNA linker. .. Free miR173 and miR173 ligated to the RNA linker were resolved by gel electrophoresis and detected by RNA filter hybridization using a probe complementary to miR173.

Methylation:

Article Title: An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation
Article Snippet: First, the template was assembled by polymerase chain reaction using the 5′-half sense primer (5′-GCGTAATACGACTCACTATAGCGCGGATAGCTCAGTCG-3′) and the 5′-half antisense primer (5′-TCAATCCCCTGCTCTACCGACTGAGCTATCCG-3′); second, the product of this assembly was further amplified using the sense T7 promoter primer (5′-GCGTAATACGACTCACTATAG-3′) and a methylated reverse primer (5′-mUmCAATCCCCTGCTCTACCGAC-3′). .. To generate full-length 2AP-tRNA, 15 µM 5′-half tRNA was annealed in 1.5× excess to 2AP-3′-half tRNA in 1× T4 RNA ligase buffer (New England Biolabs) by heating at 95°C for 3 min, 65°C for 10 min and then slowly cooling from 65 to 20°C over 45 min. T4 RNA ligase (0.02 U/ml) was added to the mixture followed by incubation at 37°C for 4 h. Ligated tRNA was purified by urea-PAGE, ultraviolet-shadowing and electroelution (EluTrap electroelution system, Whatman).

Article Title: 2?-O-methylation stabilizes Piwi-associated small RNAs and ensures DNA elimination in Tetrahymena
Article Snippet: The purified scnRNAs were ligated to [5′-32 P]pCp using T4 RNA ligase (Amersham). .. The digest was mixed with synthetic 2′- O -methylated dinucleotide standards (pAmpC, pCmpC, pGmpC, and pUmpC), spotted onto HPTLC cellulose glass plates (10 × 10 cm; Merck), and resolved with isobutyric acid/25% ammonium hydroxide/water (66:1:33 by volume) in the first dimension and then with isopropanol/HCl/water (70:15:15 by volume) in the second dimension.

Mutagenesis:

Article Title: Combinatorial minimization and secondary structure determination of a nucleotide synthase ribozyme
Article Snippet: Two oligonucleotides based on the parent a15 sequence ( ) were made using 1000 Å 1 μmole columns: a1-5′-ggataatgcgcacaagtNNNNNNNNNNAATCCTGCCTATTTAAACACTAAATCTGTCGCAATGGAAACGCGGTGGCTAGGTTGCGCGTTACTACCATATTggcacctgtccacgctc; a2:-5′-ctatcccgactggcaccAGATACGCAGTAGAGTAATCGGGAAGTACTGGATGCCATAGTGTAGACAATAGGCCTCTACCAGCGGTGTGTTGTAACGCACATCGCAGCAACtgaagagcctacgac (lowercase, fixed sequence; N, random coupling; uppercase, 20% phosphoramidite mutagenesis). .. RNA was precipitated and then ligated at the 3′ end to pRpp using adenylated pRpp (AppRpp) and T4 RNA ligase (Amersham Pharmacia).

Isolation:

Article Title: Conserved features of Y RNAs: a comparison of experimentally derived secondary structures
Article Snippet: 3′-End labeling was achieved via ligating [32 P]pCp using T4 RNA ligase (Amersham Pharmacia Biotech) for 1 h at 37°C. .. After a 5 min incubation at 37°C, 0.1 mM of each (unlabeled) nucleotide was added and incubated for 1 h. Each of the labeled products was isolated from a 10% PAA/8 M urea gel.

Article Title: 2?-O-methylation stabilizes Piwi-associated small RNAs and ensures DNA elimination in Tetrahymena
Article Snippet: Total RNA was isolated with Trizol (Invitrogen) at 4 h post-mixing and separated in a preparative sequencing gel; the scnRNAs (∼26–31 nt) were purified from the gel. .. The purified scnRNAs were ligated to [5′-32 P]pCp using T4 RNA ligase (Amersham).

Article Title: Identification and characterization of new miRNAs cloned from normal mouse mammary gland
Article Snippet: Paragraph title: Small RNA isolation and cloning ... 3' adapter (5' phosphorylated) was ligated to the RNA fraction in presence of T4 RNA ligase (Amersham-Pharmacia).

Labeling:

Article Title: Conserved features of Y RNAs: a comparison of experimentally derived secondary structures
Article Snippet: .. 3′-End labeling was achieved via ligating [32 P]pCp using T4 RNA ligase (Amersham Pharmacia Biotech) for 1 h at 37°C. .. 5′-End labeling was either performed as described , or during the in vitro transcription using 50 µCi [γ-32 P]GTP.

Article Title: IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION
Article Snippet: .. Alternatively, RNAs were 3′ end labeled using T4 RNA ligase and [32 P]pCp (GE Healthcare) using the protocol supplied by the enzyme manufacturer (Ambion). .. The radiolabeled RNAs were purified by denaturing PAGE and recovered as described above.

Article Title: Facile FMR1 mRNA structure regulation by interruptions in CGG repeats
Article Snippet: .. To label the 3′ end, the RNA fragments were incubated with 20 U of T4 RNA ligase (Epicentre Technologies) and 30 μCi of [32 P]pCp (Amersham Corp.), at 4°C for 16–18 h. The labeled RNAs were purified by electrophoresis on denaturing 10% polyacrylamide gels, localized on the gels by autoradiography and recovered as described earlier ( ). .. Analysis of RNA conformation using native PAGE In order to check for structure homogeneity of the RNA molecules, all labeled transcripts were analyzed on 5–10% polyacrylamide gels (400/300/0.8 mm, acrylamide/bisakrylamide–29/1) in non-denaturing conditions.

Article Title: 2?-O-methylation stabilizes Piwi-associated small RNAs and ensures DNA elimination in Tetrahymena
Article Snippet: The purified scnRNAs were ligated to [5′-32 P]pCp using T4 RNA ligase (Amersham). .. The labeled dinucleotides were visualized by autoradiography, while the synthetic dinucleotide standards were visualized by UV shadowing.

Article Title: RISC is a 5? phosphomonoester-producing RNA endonuclease
Article Snippet: .. Site-specific internally labeled substrate RNA was prepared by incubating 2 pmol 5′-32 P-phosphorylated and linker-modified 12-nt RNA with 20 pmol 9-nt RNA, and 40 units of T4 RNA ligase (Amersham) in 10 mM MgCl2 /10 mM 2-mercaptoethanol/50 mM Tris-HCl (pH 7.6)/0.1 mg/mL acetylated bovine serum albumin (Ac-BS, Sigma)/0.2 mM ATP/15% DMSO in a final volume of 20 μL. ..

Article Title: MicroRNAs Regulate Human Brain Endothelial Cell-Barrier Function in Inflammation: Implications for Multiple Sclerosis
Article Snippet: .. Total RNA (100 ng) was labeled with pCp-Cy3 using T4 RNA ligase (GE Healthcare) and the Agilent human microRNA microarray kit annotated in the Sanger miRbase (release 12.0) containing probes for 939 human microRNAs was performed according to the Agilent's microRNA microarray profiling. .. GeneSpring GX 9 software (Agilent Technologies) was used for value extraction.

Purification:

Article Title: An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation
Article Snippet: .. To generate full-length 2AP-tRNA, 15 µM 5′-half tRNA was annealed in 1.5× excess to 2AP-3′-half tRNA in 1× T4 RNA ligase buffer (New England Biolabs) by heating at 95°C for 3 min, 65°C for 10 min and then slowly cooling from 65 to 20°C over 45 min. T4 RNA ligase (0.02 U/ml) was added to the mixture followed by incubation at 37°C for 4 h. Ligated tRNA was purified by urea-PAGE, ultraviolet-shadowing and electroelution (EluTrap electroelution system, Whatman). .. Fluorescence spectroscopy and stopped-flow experiments 2AP-tRNA (0.05–0.2 µM) diluted in 1× TAKEM4 buffer was excited at 325 nm, titrated with TruB and the emission was measured from 340 to 400 nm using a Quanta Master 60 fluorescence spectrometer (Photon Technology International).

Article Title: IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION
Article Snippet: Alternatively, RNAs were 3′ end labeled using T4 RNA ligase and [32 P]pCp (GE Healthcare) using the protocol supplied by the enzyme manufacturer (Ambion). .. The radiolabeled RNAs were purified by denaturing PAGE and recovered as described above.

Article Title: Facile FMR1 mRNA structure regulation by interruptions in CGG repeats
Article Snippet: .. To label the 3′ end, the RNA fragments were incubated with 20 U of T4 RNA ligase (Epicentre Technologies) and 30 μCi of [32 P]pCp (Amersham Corp.), at 4°C for 16–18 h. The labeled RNAs were purified by electrophoresis on denaturing 10% polyacrylamide gels, localized on the gels by autoradiography and recovered as described earlier ( ). .. Analysis of RNA conformation using native PAGE In order to check for structure homogeneity of the RNA molecules, all labeled transcripts were analyzed on 5–10% polyacrylamide gels (400/300/0.8 mm, acrylamide/bisakrylamide–29/1) in non-denaturing conditions.

Article Title: 2?-O-methylation stabilizes Piwi-associated small RNAs and ensures DNA elimination in Tetrahymena
Article Snippet: .. The purified scnRNAs were ligated to [5′-32 P]pCp using T4 RNA ligase (Amersham). .. The ligated scnRNAs–pCp were gel purified and digested for 20 min at 37°C using P1 nuclease (400 mU/mL final) (Sigma).

Sequencing:

Article Title: 2?-O-methylation stabilizes Piwi-associated small RNAs and ensures DNA elimination in Tetrahymena
Article Snippet: Total RNA was isolated with Trizol (Invitrogen) at 4 h post-mixing and separated in a preparative sequencing gel; the scnRNAs (∼26–31 nt) were purified from the gel. .. The purified scnRNAs were ligated to [5′-32 P]pCp using T4 RNA ligase (Amersham).

Article Title: Genomic Characterization of a Novel Virus of the Family Tymoviridae Isolated from Mosquitoes
Article Snippet: Paragraph title: Genome sequencing ... Briefly, the 3′-end of the viral RNA was ligated with an adapter (5′-P-GTCGATCACGCGATCGAACGGTCGC TGAG-3′) using T4 RNA ligase (Amersham).

Article Title: Identification and characterization of new miRNAs cloned from normal mouse mammary gland
Article Snippet: 3' adapter (5' phosphorylated) was ligated to the RNA fraction in presence of T4 RNA ligase (Amersham-Pharmacia). .. The sample was again size fractionated using 15% PAGE before to be reverse transcribed using primer complementary to the 3' adapter sequence and PCR amplified using primers on both adapters.

Article Title: Combinatorial minimization and secondary structure determination of a nucleotide synthase ribozyme
Article Snippet: Two oligonucleotides based on the parent a15 sequence ( ) were made using 1000 Å 1 μmole columns: a1-5′-ggataatgcgcacaagtNNNNNNNNNNAATCCTGCCTATTTAAACACTAAATCTGTCGCAATGGAAACGCGGTGGCTAGGTTGCGCGTTACTACCATATTggcacctgtccacgctc; a2:-5′-ctatcccgactggcaccAGATACGCAGTAGAGTAATCGGGAAGTACTGGATGCCATAGTGTAGACAATAGGCCTCTACCAGCGGTGTGTTGTAACGCACATCGCAGCAACtgaagagcctacgac (lowercase, fixed sequence; N, random coupling; uppercase, 20% phosphoramidite mutagenesis). .. RNA was precipitated and then ligated at the 3′ end to pRpp using adenylated pRpp (AppRpp) and T4 RNA ligase (Amersham Pharmacia).

Immunoprecipitation:

Article Title: Physical and Functional Interactions of the Arf Tumor Suppressor Protein with Nucleophosmin/B23
Article Snippet: Paragraph title: Immunoprecipitation, immunoblotting, and RNA end labeling. ... RNAs were recovered by phenol-chloroform extraction followed by ethanol precipitation in 0.3 M sodium acetate containing 20 μg of glycogen (Roche, Basel, Switzerland), and end-labeled with 40 μCi of [32 P]cytidine 3′, 5′-bis(phosphate) ([32 ]pCp) (3,000 Ci/mmol; ICN, Costa Mesa, Calif.) with T4 RNA ligase (Amersham Biosciences) as described ( ).

Polyacrylamide Gel Electrophoresis:

Article Title: IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION
Article Snippet: Alternatively, RNAs were 3′ end labeled using T4 RNA ligase and [32 P]pCp (GE Healthcare) using the protocol supplied by the enzyme manufacturer (Ambion). .. The radiolabeled RNAs were purified by denaturing PAGE and recovered as described above.

Article Title: Identification and characterization of new miRNAs cloned from normal mouse mammary gland
Article Snippet: Briefly, 500 μg of RNA were size fractionated using 15% denaturing polyacrylamide gel electrophoresis (PAGE). .. 3' adapter (5' phosphorylated) was ligated to the RNA fraction in presence of T4 RNA ligase (Amersham-Pharmacia).

Nested PCR:

Article Title: Global analysis of the mammalian RNA degradome reveals widespread miRNA-dependent and miRNA-independent endonucleolytic cleavage
Article Snippet: Gene specific 5′-RACE For the identification of RISC, 0.3 µg of 5′-RACE adapter (Ambion First Choice RLM-RACE kit; Applied Biosystems) was ligated to 2 µg DNase I-treated total RNA from human rectal mucosa, overnight at 14°C, using T4 RNA Ligase (GE Life Sciences). .. Touch-down PCR conditions were 94°C for 30 s, 60–50°C for 30 s, 72°C for 1 min, over 20 cycles, then 30 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 1 min. To enrich for cleaved sequences, one fiftieth of the touchdown reaction was then used as template in a second ‘nested PCR’ reaction containing 0.2 µM 5′RACE Inner Primer (FirstChoice® RLM-RACE kit; Applied Biosystems; 5′-CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG-3′) and 0.2 µM RICS Nested Primer (5′-AAGCTTCCTGCTCCTCAGGCTG-3′) with 40 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 1 min. For the identification of PDK2 and Tspan18, the above procedure was employed using 1 µg of poly (A+) RNA ligated to the 5′ adaptor (5′-GUUCAGAGUUCUACAGUCCGAC-3′).

Rapid Amplification of cDNA Ends:

Article Title: NtcA-Regulated Heterocyst Differentiation Genes hetC and devB from Anabaena sp. Strain PCC 7120 Exhibit a Similar Tandem Promoter Arrangement
Article Snippet: Paragraph title: 5′ rapid amplification of cDNA ends (RACE). ... Sixty picomoles of 5′ RNA adaptor (Table ) (Sigma Proligo) was added to the RNA sample, and ligation was performed in the presence of 80 units of T4 RNA ligase (Amersham) for 16 h at 15°C.

Plasmid Preparation:

Article Title: Identification and characterization of new miRNAs cloned from normal mouse mammary gland
Article Snippet: 3' adapter (5' phosphorylated) was ligated to the RNA fraction in presence of T4 RNA ligase (Amersham-Pharmacia). .. Amplified products were cloned using pGEM-T vector system I (Promega).

Software:

Article Title: MicroRNAs Regulate Human Brain Endothelial Cell-Barrier Function in Inflammation: Implications for Multiple Sclerosis
Article Snippet: Total RNA (100 ng) was labeled with pCp-Cy3 using T4 RNA ligase (GE Healthcare) and the Agilent human microRNA microarray kit annotated in the Sanger miRbase (release 12.0) containing probes for 939 human microRNAs was performed according to the Agilent's microRNA microarray profiling. .. GeneSpring GX 9 software (Agilent Technologies) was used for value extraction.

Selection:

Article Title: Combinatorial minimization and secondary structure determination of a nucleotide synthase ribozyme
Article Snippet: Paragraph title: Degenerate pool synthesis and selection protocol ... RNA was precipitated and then ligated at the 3′ end to pRpp using adenylated pRpp (AppRpp) and T4 RNA ligase (Amersham Pharmacia).

In Vitro:

Article Title: Conserved features of Y RNAs: a comparison of experimentally derived secondary structures
Article Snippet: 3′-End labeling was achieved via ligating [32 P]pCp using T4 RNA ligase (Amersham Pharmacia Biotech) for 1 h at 37°C. .. 5′-End labeling was either performed as described , or during the in vitro transcription using 50 µCi [γ-32 P]GTP.

Article Title: An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation
Article Snippet: The 5′-half tRNA template was then used in 2 ml in vitro transcription reactions using the same procedure as outlined for [3 H]-tRNA, but including 3 mM nonradioactive UTP. .. To generate full-length 2AP-tRNA, 15 µM 5′-half tRNA was annealed in 1.5× excess to 2AP-3′-half tRNA in 1× T4 RNA ligase buffer (New England Biolabs) by heating at 95°C for 3 min, 65°C for 10 min and then slowly cooling from 65 to 20°C over 45 min. T4 RNA ligase (0.02 U/ml) was added to the mixture followed by incubation at 37°C for 4 h. Ligated tRNA was purified by urea-PAGE, ultraviolet-shadowing and electroelution (EluTrap electroelution system, Whatman).

Article Title: IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION
Article Snippet: Internally 32 P-labeled RNAs were produced by in vitro transcription of the resulting double-stranded DNAs in the presence of [α-32 P]ATP. .. Alternatively, RNAs were 3′ end labeled using T4 RNA ligase and [32 P]pCp (GE Healthcare) using the protocol supplied by the enzyme manufacturer (Ambion).

Article Title: Facile FMR1 mRNA structure regulation by interruptions in CGG repeats
Article Snippet: Paragraph title: In vitro transcription and RNA labeling ... To label the 3′ end, the RNA fragments were incubated with 20 U of T4 RNA ligase (Epicentre Technologies) and 30 μCi of [32 P]pCp (Amersham Corp.), at 4°C for 16–18 h. The labeled RNAs were purified by electrophoresis on denaturing 10% polyacrylamide gels, localized on the gels by autoradiography and recovered as described earlier ( ).

Ethanol Precipitation:

Article Title: RNA-Dependent DNA Binding Activity of the Pur Factor, Potentially Involved in DNA Replication and Gene Transcription
Article Snippet: .. After phenol extraction and ethanol precipitation in the presence of 1 μ g glycogen, RNAs were ligated to [32 P]Cp (100 μ Ci, Amersham) by the T4 RNA ligase (20 U) (Biolabs) in 50 mM Tris-Cl− (pH 7.8), 10 mM MgCl2 , 10 mM β -mercaptoethanol, 1 mM ATP, and 1 mM hexamine cobalt chloride, in the presence of 20 U RNasin (Promega) in a final volume of 30 μ l at 10°C for 16 h. Aliquots (10 μ l) of the ligation reactions were mixed to an equal volume of formamide buffer and incubated at 90 °C for 5 min prior to electrophoresis. .. The 1.1-kb-long Purα full-length cDNA present in the insert of the λgt11 clone λAB6 , kindly provided by Dr. Johnson, was subcloned into the EcoRI site of the pBSK plasmid.

Article Title: An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation
Article Snippet: Following ethanol precipitation, the RNA was purified using the BioRad Model 491 Prep Cell continuous electrophoresis purification system. .. To generate full-length 2AP-tRNA, 15 µM 5′-half tRNA was annealed in 1.5× excess to 2AP-3′-half tRNA in 1× T4 RNA ligase buffer (New England Biolabs) by heating at 95°C for 3 min, 65°C for 10 min and then slowly cooling from 65 to 20°C over 45 min. T4 RNA ligase (0.02 U/ml) was added to the mixture followed by incubation at 37°C for 4 h. Ligated tRNA was purified by urea-PAGE, ultraviolet-shadowing and electroelution (EluTrap electroelution system, Whatman).

Article Title: Physical and Functional Interactions of the Arf Tumor Suppressor Protein with Nucleophosmin/B23
Article Snippet: .. RNAs were recovered by phenol-chloroform extraction followed by ethanol precipitation in 0.3 M sodium acetate containing 20 μg of glycogen (Roche, Basel, Switzerland), and end-labeled with 40 μCi of [32 P]cytidine 3′, 5′-bis(phosphate) ([32 ]pCp) (3,000 Ci/mmol; ICN, Costa Mesa, Calif.) with T4 RNA ligase (Amersham Biosciences) as described ( ). .. Cells were metabolically labeled with 2.5 μCi of [3 H]uridine per ml (30 to 60 Ci/mmol, Amersham Biosciences) in complete medium for 30 min, washed twice with PBS, and incubated in complete medium without the radioactive precursor for 2 h. Total RNAs were isolated with Trizol (Invitrogen, Carlsbad, Calif.), and quantitated by absorbance measurement, and their specific activities were determined by liquid scintillation.

High Performance Thin Layer Chromatography:

Article Title: 2?-O-methylation stabilizes Piwi-associated small RNAs and ensures DNA elimination in Tetrahymena
Article Snippet: The purified scnRNAs were ligated to [5′-32 P]pCp using T4 RNA ligase (Amersham). .. The digest was mixed with synthetic 2′- O -methylated dinucleotide standards (pAmpC, pCmpC, pGmpC, and pUmpC), spotted onto HPTLC cellulose glass plates (10 × 10 cm; Merck), and resolved with isobutyric acid/25% ammonium hydroxide/water (66:1:33 by volume) in the first dimension and then with isopropanol/HCl/water (70:15:15 by volume) in the second dimension.

Spectrophotometry:

Article Title: MicroRNAs Regulate Human Brain Endothelial Cell-Barrier Function in Inflammation: Implications for Multiple Sclerosis
Article Snippet: The quantity (NanoDrop 1000 spectrophotometer) and the quality (2100 Bioanalyzer, RNA 6000 Pico LabChip; Agilent Technologies) were assessed for each sample. .. Total RNA (100 ng) was labeled with pCp-Cy3 using T4 RNA ligase (GE Healthcare) and the Agilent human microRNA microarray kit annotated in the Sanger miRbase (release 12.0) containing probes for 939 human microRNAs was performed according to the Agilent's microRNA microarray profiling.

Produced:

Article Title: IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION
Article Snippet: Internally 32 P-labeled RNAs were produced by in vitro transcription of the resulting double-stranded DNAs in the presence of [α-32 P]ATP. .. Alternatively, RNAs were 3′ end labeled using T4 RNA ligase and [32 P]pCp (GE Healthcare) using the protocol supplied by the enzyme manufacturer (Ambion).

FLAG-tag:

Article Title: Physical and Functional Interactions of the Arf Tumor Suppressor Protein with Nucleophosmin/B23
Article Snippet: Proteins were detected with antibodies to p19Arf , p53 (Ab-7, Oncogene Research Products, San Diego, Calif.), p21Cip1 (C-19, Santa Cruz Biotechnology, Santa Cruz, Calif.), NPM (C-19 Santa Cruz), Flag tag (M2, Sigma Chemicals), or ribosomal protein L7 (Novus Biologicals, NB200-308). .. RNAs were recovered by phenol-chloroform extraction followed by ethanol precipitation in 0.3 M sodium acetate containing 20 μg of glycogen (Roche, Basel, Switzerland), and end-labeled with 40 μCi of [32 P]cytidine 3′, 5′-bis(phosphate) ([32 ]pCp) (3,000 Ci/mmol; ICN, Costa Mesa, Calif.) with T4 RNA ligase (Amersham Biosciences) as described ( ).

Thin Layer Chromatography:

Article Title: 2?-O-methylation stabilizes Piwi-associated small RNAs and ensures DNA elimination in Tetrahymena
Article Snippet: Paragraph title: Two-dimensional thin-layer chromatography ... The purified scnRNAs were ligated to [5′-32 P]pCp using T4 RNA ligase (Amersham).

End Labeling:

Article Title: RNA-Dependent DNA Binding Activity of the Pur Factor, Potentially Involved in DNA Replication and Gene Transcription
Article Snippet: Paragraph title: 3′ End Labeling of RNAs ... After phenol extraction and ethanol precipitation in the presence of 1 μ g glycogen, RNAs were ligated to [32 P]Cp (100 μ Ci, Amersham) by the T4 RNA ligase (20 U) (Biolabs) in 50 mM Tris-Cl− (pH 7.8), 10 mM MgCl2 , 10 mM β -mercaptoethanol, 1 mM ATP, and 1 mM hexamine cobalt chloride, in the presence of 20 U RNasin (Promega) in a final volume of 30 μ l at 10°C for 16 h. Aliquots (10 μ l) of the ligation reactions were mixed to an equal volume of formamide buffer and incubated at 90 °C for 5 min prior to electrophoresis.

Article Title: Facile FMR1 mRNA structure regulation by interruptions in CGG repeats
Article Snippet: In vitro transcription and RNA labeling In vitro transcription reactions and 5′ end labeling of RNA molecules were performed as described earlier ( ). .. To label the 3′ end, the RNA fragments were incubated with 20 U of T4 RNA ligase (Epicentre Technologies) and 30 μCi of [32 P]pCp (Amersham Corp.), at 4°C for 16–18 h. The labeled RNAs were purified by electrophoresis on denaturing 10% polyacrylamide gels, localized on the gels by autoradiography and recovered as described earlier ( ).

Article Title: Physical and Functional Interactions of the Arf Tumor Suppressor Protein with Nucleophosmin/B23
Article Snippet: Paragraph title: Immunoprecipitation, immunoblotting, and RNA end labeling. ... RNAs were recovered by phenol-chloroform extraction followed by ethanol precipitation in 0.3 M sodium acetate containing 20 μg of glycogen (Roche, Basel, Switzerland), and end-labeled with 40 μCi of [32 P]cytidine 3′, 5′-bis(phosphate) ([32 ]pCp) (3,000 Ci/mmol; ICN, Costa Mesa, Calif.) with T4 RNA ligase (Amersham Biosciences) as described ( ).

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    GE Healthcare t4 rna ligase
    RNA-DNA ligation with <t>T4</t> RNA ligase. ( A ) The 21- to 24-nt smRNAs from infected HC-Pro + or HC-Pro - plants (lanes 5-12) or the 24-nt smRNAs from both infected and uninfected HC-Pro + and HC-Pro - plants (lanes 13-28) were gel-purified and ligated to a 16-nt
    T4 Rna Ligase, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase/product/GE Healthcare
    Average 90 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase - by Bioz Stars, 2020-02
    90/100 stars
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    RNA-DNA ligation with T4 RNA ligase. ( A ) The 21- to 24-nt smRNAs from infected HC-Pro + or HC-Pro - plants (lanes 5-12) or the 24-nt smRNAs from both infected and uninfected HC-Pro + and HC-Pro - plants (lanes 13-28) were gel-purified and ligated to a 16-nt

    Journal:

    Article Title: Extensive 3? modification of plant small RNAs is modulated by helper component-proteinase expression

    doi: 10.1073/pnas.0506597102

    Figure Lengend Snippet: RNA-DNA ligation with T4 RNA ligase. ( A ) The 21- to 24-nt smRNAs from infected HC-Pro + or HC-Pro - plants (lanes 5-12) or the 24-nt smRNAs from both infected and uninfected HC-Pro + and HC-Pro - plants (lanes 13-28) were gel-purified and ligated to a 16-nt

    Article Snippet: Radiolabeled 21- to 24-nt smRNAs from virus-infected HC-Pro+ or HC-Pro- plants, together with the four gel-purified samples of 24-nt smRNAs (the same as used in ), were tested for their ability to serve as substrates for T4 RNA ligase.

    Techniques: DNA Ligation, Infection, Purification