t4 rna ligase buffer  (TaKaRa)

 
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    Name:
    T4 RNA Ligase
    Description:
    T4 RNA Ligase is a ligation enzyme for joining single stranded RNA or DNA fragments by catalyzing the formation of phosphodiester bonds between single stranded RNA or DNA containing 5 phosphate and 3 hydroxyl termini This enzyme requires ATP for activity and its activity is highest for RNA RNA ligation lower for RNA DNA ligation and lowest for DNA DNA ligation 3 end labeling is often completed using 5 32P pCp
    Catalog Number:
    2050b
    Price:
    None
    Size:
    5 000 Units
    Category:
    T4 RNA Ligase Ligation enzymes Cloning
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    Structured Review

    TaKaRa t4 rna ligase buffer
    Gel electrophoresis pattern of mRNA-linker ligation. The ligation products reacted with or without prRT- DNA oligomer used as a blocker of the 3'-end of mRNA were electrophoresis on 8 M urea 8 % PAGE at 65 °C and were visualized with fluorescence of (A) SYBR Green II and (B) FITC. Lane M: DNA ladder, Lane Y: ligation product, Lane L-: negative control, reaction product without DNA-linker, Lane E-: negative control, reaction product without <t>T4</t> RNA ligase. Mobility of the mRNA-linker and the self-ligation product of mRNA are shown to be equivalent.
    T4 RNA Ligase is a ligation enzyme for joining single stranded RNA or DNA fragments by catalyzing the formation of phosphodiester bonds between single stranded RNA or DNA containing 5 phosphate and 3 hydroxyl termini This enzyme requires ATP for activity and its activity is highest for RNA RNA ligation lower for RNA DNA ligation and lowest for DNA DNA ligation 3 end labeling is often completed using 5 32P pCp
    https://www.bioz.com/result/t4 rna ligase buffer/product/TaKaRa
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    Price from $9.99 to $1999.99
    t4 rna ligase buffer - by Bioz Stars, 2020-03
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    Images

    1) Product Images from "An mRNA-protein Fusion at N-terminus for Evolutionary Protein Engineering"

    Article Title: An mRNA-protein Fusion at N-terminus for Evolutionary Protein Engineering

    Journal: International Journal of Biological Sciences

    doi:

    Gel electrophoresis pattern of mRNA-linker ligation. The ligation products reacted with or without prRT- DNA oligomer used as a blocker of the 3'-end of mRNA were electrophoresis on 8 M urea 8 % PAGE at 65 °C and were visualized with fluorescence of (A) SYBR Green II and (B) FITC. Lane M: DNA ladder, Lane Y: ligation product, Lane L-: negative control, reaction product without DNA-linker, Lane E-: negative control, reaction product without T4 RNA ligase. Mobility of the mRNA-linker and the self-ligation product of mRNA are shown to be equivalent.
    Figure Legend Snippet: Gel electrophoresis pattern of mRNA-linker ligation. The ligation products reacted with or without prRT- DNA oligomer used as a blocker of the 3'-end of mRNA were electrophoresis on 8 M urea 8 % PAGE at 65 °C and were visualized with fluorescence of (A) SYBR Green II and (B) FITC. Lane M: DNA ladder, Lane Y: ligation product, Lane L-: negative control, reaction product without DNA-linker, Lane E-: negative control, reaction product without T4 RNA ligase. Mobility of the mRNA-linker and the self-ligation product of mRNA are shown to be equivalent.

    Techniques Used: Nucleic Acid Electrophoresis, Ligation, Electrophoresis, Polyacrylamide Gel Electrophoresis, Fluorescence, SYBR Green Assay, Negative Control

    Screening cycle of the mRNA-protein fusion in this study. The dsDNA library is transcribed to mRNA. The mRNA is hybridized to the DNA moiety of the linker having hydrazide group and ligated with T4 RNA ligase. Hydrazide group of the ligated product and acetyl group of the phenylalanine derivative that is acylated to sup tRNA are ligated chemically and the modified mRNA is translated. The modified aminoacyl sup tRNA tends to occupy the A-site of ribosome at UAG codon inserted near downstream of initiation codon and the phenylalanine is incorporated into the growing peptide. Thus, linkage between N-terminus of the nascent peptide and 5'-terminus of its mRNA is achieved. Screening of mRNA-peptide fusion library according to property of the displayed peptide and amplify the genotype molecules of the screened fusions by RT-PCR.
    Figure Legend Snippet: Screening cycle of the mRNA-protein fusion in this study. The dsDNA library is transcribed to mRNA. The mRNA is hybridized to the DNA moiety of the linker having hydrazide group and ligated with T4 RNA ligase. Hydrazide group of the ligated product and acetyl group of the phenylalanine derivative that is acylated to sup tRNA are ligated chemically and the modified mRNA is translated. The modified aminoacyl sup tRNA tends to occupy the A-site of ribosome at UAG codon inserted near downstream of initiation codon and the phenylalanine is incorporated into the growing peptide. Thus, linkage between N-terminus of the nascent peptide and 5'-terminus of its mRNA is achieved. Screening of mRNA-peptide fusion library according to property of the displayed peptide and amplify the genotype molecules of the screened fusions by RT-PCR.

    Techniques Used: Modification, Reverse Transcription Polymerase Chain Reaction

    Related Articles

    Clone Assay:

    Article Title: Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells
    Article Snippet: The lysates were then incubated with T4 RNA ligase (TAKARA) overnight at 37°C to induce intra-molecular DNA joining and produce circular VSV DNA ( ). .. Nested PCR was performed using the N1228-F/N163-R primer pair, and the nested PCR products were cloned into the pGEM®-T Easy vector and sequenced using the SP6 and T7 promoter primers.

    Article Title: The expression profile of microRNAs in mouse embryos
    Article Snippet: Ligation was carried out using T4 RNA ligase (Takara Bio Inc.) at 15°C for 15 h. RNA fraction with attached adapters was purified using PAGE gel, phosphorylated by T4 polynucleotide kinase (Takara Bio Inc.) and followed by second round of ligation with the DNA–RNA chimeric 5′-adaptor containing the GAUC site (5′-CCATGTTCGCATCGGCaggauc-3′, RNA is shown in lowercase). .. PCR products were purified, digested by SfaNI (NEB) and cloned into the Tag vector pMBS I (Solexa), linearized with BamHI and BbsI.

    Amplification:

    Article Title: The expression profile of microRNAs in mouse embryos
    Article Snippet: Ligation was carried out using T4 RNA ligase (Takara Bio Inc.) at 15°C for 15 h. RNA fraction with attached adapters was purified using PAGE gel, phosphorylated by T4 polynucleotide kinase (Takara Bio Inc.) and followed by second round of ligation with the DNA–RNA chimeric 5′-adaptor containing the GAUC site (5′-CCATGTTCGCATCGGCaggauc-3′, RNA is shown in lowercase). .. The cDNA was amplified by 12 cycles of PCR using Pyrobest DNA Polymerase (Takara Bio Inc.) and PCR primers (5′-CCATGTTCGCATCGGCAGGATC-3′, 5′-AGCCGTCAGCATCATTGAGCAT-3′) in the presence of 5-methylated-dCTP, dATP, dGTP, dTTP mixture.

    Synthesized:

    Article Title: Easy and Rapid Binding Assay for Functional Analysis of Disulfide-Containing Peptides by a Pull-Down Method Using a Puromycin-Linker and a Cell-Free Translation System
    Article Snippet: Biotin-attached peptide was prepared as follows: DNA was transcribed to mRNA using the T7 RiboMAX Express Large Scale RNA Production System (Promega, Madison, WI, USA), and the synthesized mRNA was purified with an After Tri Reagent RNA Clean-up Kit (Favorgen, Ping-Tung, Taiwan). .. The puromycin-linker was hybridized to the purified mRNA, and the 5'-terminus of the puromycin-linker and the 3'-terminus of the mRNA were ligated with T4 RNA ligase (Takara Bio, Otsu, Japan) and polynucleotide kinase (PNK; Takara Bio) at 25 °C for 1 h. Six picomoles of the ligation product were placed in 50 µL of a cell-free translation reaction solution with the Retic Lysate IVT kit (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 30 °C for 30 min. Then, 20 µL of 3 M KCl and 6 µL of 1 M MgCl2 were added to the reaction solution and incubated at 37 °C for 1 h. Eighteen microliters of EDTA solution (0.5 M, pH 8.0) were added to the translation reaction and incubated at 25 °C for 10 min to remove bound ribosomes.

    Construct:

    Article Title: Easy and Rapid Binding Assay for Functional Analysis of Disulfide-Containing Peptides by a Pull-Down Method Using a Puromycin-Linker and a Cell-Free Translation System
    Article Snippet: Pull-Down Method for Disulfide-Containing Peptides A schematic of the pull-down method and the puromycin-linker construct is shown in . .. The puromycin-linker was hybridized to the purified mRNA, and the 5'-terminus of the puromycin-linker and the 3'-terminus of the mRNA were ligated with T4 RNA ligase (Takara Bio, Otsu, Japan) and polynucleotide kinase (PNK; Takara Bio) at 25 °C for 1 h. Six picomoles of the ligation product were placed in 50 µL of a cell-free translation reaction solution with the Retic Lysate IVT kit (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 30 °C for 30 min. Then, 20 µL of 3 M KCl and 6 µL of 1 M MgCl2 were added to the reaction solution and incubated at 37 °C for 1 h. Eighteen microliters of EDTA solution (0.5 M, pH 8.0) were added to the translation reaction and incubated at 25 °C for 10 min to remove bound ribosomes.

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
    Article Snippet: The construct of genome has a T7 promoter, a translational enhancer of TMV 5' UTR , an initiation codon with good context , a coding sequence of His-tag , an N-terminus sequence in the GFP gene and a GC-clump sequence for hybridization to a GC-rich DNA-oligomer. .. The ligation reaction with T4 RNA ligase (1 U/µl, TAKARA) was performed in the supplier's buffer with 10% DMSO at 25 ºC.

    SYBR Green Assay:

    Article Title: An mRNA-protein Fusion at N-terminus for Evolutionary Protein Engineering
    Article Snippet: Ligation between the mRNA and the hydrazide-linker The mRNA (2 µM) was hybridized to the DNA moiety of the hydrazide-linker (4 µM) and prRT- (4 µM) by heating at 95 °C and cooling to 25 °C in 50 µl of T4 RNA ligase buffer (Takara Bio) and ligation reaction was started by adding T4 RNA ligase (40 U, Takara Bio) and ribonuclease inhibitor (40 U, Takara Bio). .. Ligation reaction was performed at 25 °C for overnight and the ligated product was analyzed by 8 M urea 8 % PAGE using TBE running buffer at 65 °C and were visualized with fluorescence of FITC and then visualized again after staining by SYBR Green II (Cambrex) using a fluorescence imager (Pharos FX; Bio-Rad).

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
    Article Snippet: Ligate the mRNA with the hybridized Linker-Y using T4 RNA ligase (1 U/µl, TAKARA) in the supplier's buffer with 10% DMSO for 15 min at 25 ºC. .. Check the FITC fluorescent gel bands using a fluorescence imager (Molecular Imager FX; BIO-RAD), and then check again after staining by SYBR Green II (FMC).

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
    Article Snippet: The ligation reaction with T4 RNA ligase (1 U/µl, TAKARA) was performed in the supplier's buffer with 10% DMSO at 25 ºC. .. The ligation products were analyzed by 8 M urea 10% PAGE using TBE running buffer at 65 ºC and were visualized with fluorescence of FITC and then visualized again after staining by SYBR Green II (FMC) using an fluorescence imager (Molecular Imager FX; BIO-RAD).

    cDNA Library Assay:

    Article Title: The expression profile of microRNAs in mouse embryos
    Article Snippet: Paragraph title: Samples preparation and cDNA library construction ... Ligation was carried out using T4 RNA ligase (Takara Bio Inc.) at 15°C for 15 h. RNA fraction with attached adapters was purified using PAGE gel, phosphorylated by T4 polynucleotide kinase (Takara Bio Inc.) and followed by second round of ligation with the DNA–RNA chimeric 5′-adaptor containing the GAUC site (5′-CCATGTTCGCATCGGCaggauc-3′, RNA is shown in lowercase).

    Incubation:

    Article Title: Easy and Rapid Binding Assay for Functional Analysis of Disulfide-Containing Peptides by a Pull-Down Method Using a Puromycin-Linker and a Cell-Free Translation System
    Article Snippet: .. The puromycin-linker was hybridized to the purified mRNA, and the 5'-terminus of the puromycin-linker and the 3'-terminus of the mRNA were ligated with T4 RNA ligase (Takara Bio, Otsu, Japan) and polynucleotide kinase (PNK; Takara Bio) at 25 °C for 1 h. Six picomoles of the ligation product were placed in 50 µL of a cell-free translation reaction solution with the Retic Lysate IVT kit (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 30 °C for 30 min. Then, 20 µL of 3 M KCl and 6 µL of 1 M MgCl2 were added to the reaction solution and incubated at 37 °C for 1 h. Eighteen microliters of EDTA solution (0.5 M, pH 8.0) were added to the translation reaction and incubated at 25 °C for 10 min to remove bound ribosomes. .. The mRNA-linker-peptide fusion molecules were isolated from the translation reaction solution using 30 μL of Dynabeads MyOne Streptavidin C1 (SA-beads; Thermo Fisher Scientific) according to the supplier’s instructions.

    Article Title: Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells
    Article Snippet: .. The lysates were then incubated with T4 RNA ligase (TAKARA) overnight at 37°C to induce intra-molecular DNA joining and produce circular VSV DNA ( ). .. The ligated DNA was purified using phenol/chloroform and subjected to PCR with the N1173-F/N681-R primer pair.

    Activity Assay:

    Article Title: Efficacy of a Novel Class of RNA Interference Therapeutic Agents
    Article Snippet: The RNA 5′ end was phosphorylated by treating with T4 polynucleotide kinase (TaKaRa) in a reaction buffer containing 50 pmol RNA, enzyme 20 Units, 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2 , 5 mM DTT and 1 mM ATP at 37°C for 2 h. After phenol/chloroform extraction and ethanol precipitation, the RNA 3′ and 5′ ends were ligated and the RNA converted to a circular type by treating overnight with T4 RNA ligase (TaKaRa) at 16°C in a reaction buffer containing RNA 2 ug, Enzyme 50 U, 50 mM Tris-HCl, pH7.5, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP, 0.006% BSA. .. The inhibitory activity of dumbbell RNA directed against human GAPDH was evaluated and compared with nkRNA in HCT 116 cell lines.

    Mass Spectrometry:

    Article Title: Efficacy of a Novel Class of RNA Interference Therapeutic Agents
    Article Snippet: The RNA 5′ end was phosphorylated by treating with T4 polynucleotide kinase (TaKaRa) in a reaction buffer containing 50 pmol RNA, enzyme 20 Units, 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2 , 5 mM DTT and 1 mM ATP at 37°C for 2 h. After phenol/chloroform extraction and ethanol precipitation, the RNA 3′ and 5′ ends were ligated and the RNA converted to a circular type by treating overnight with T4 RNA ligase (TaKaRa) at 16°C in a reaction buffer containing RNA 2 ug, Enzyme 50 U, 50 mM Tris-HCl, pH7.5, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP, 0.006% BSA. .. After phenol/chloroform extraction and ethanol precipitation, the formation of RNA was confirmed in 7 M urea-denatured 15% PAGE and by ESI-Q-TOF mass spectrometry.

    BIA-KA:

    Article Title: In vitro selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in an Escherichia coli cell-free translation system
    Article Snippet: .. T4 RNA ligase, Bca BEST RNA PCR kit ver1.1, GelStar Nucleic Acid Stain and ribonuclease inhibitor were from TaKaRa BIO. .. Escherichia coli S30 Extract System for Linear Templates and pGEM-T vector were from Promega.

    Modification:

    Article Title: The expression profile of microRNAs in mouse embryos
    Article Snippet: Samples preparation and cDNA library construction The method for cDNA library construction for the MPSS analysis was modified and is shown in . .. Ligation was carried out using T4 RNA ligase (Takara Bio Inc.) at 15°C for 15 h. RNA fraction with attached adapters was purified using PAGE gel, phosphorylated by T4 polynucleotide kinase (Takara Bio Inc.) and followed by second round of ligation with the DNA–RNA chimeric 5′-adaptor containing the GAUC site (5′-CCATGTTCGCATCGGCaggauc-3′, RNA is shown in lowercase).

    Hybridization:

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
    Article Snippet: The construct of genome has a T7 promoter, a translational enhancer of TMV 5' UTR , an initiation codon with good context , a coding sequence of His-tag , an N-terminus sequence in the GFP gene and a GC-clump sequence for hybridization to a GC-rich DNA-oligomer. .. The ligation reaction with T4 RNA ligase (1 U/µl, TAKARA) was performed in the supplier's buffer with 10% DMSO at 25 ºC.

    Inverse PCR:

    Article Title: Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells
    Article Snippet: Paragraph title: Inverse PCR to determine the end structures of VSV DNA ... The lysates were then incubated with T4 RNA ligase (TAKARA) overnight at 37°C to induce intra-molecular DNA joining and produce circular VSV DNA ( ).

    Ligation:

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
    Article Snippet: .. Ligation reaction between mRNA and puromycin-linker We compared three puromycin-linker attachment methods, which are the splint ligation with T4 DNA ligase , the splint ligation with T4 RNA ligase and the Y-ligation with T4 RNA ligase (Fig. ). .. The splint ligation method with T4 DNA ligase gave a low product yield because of undesirable mRNA terminal heterogeneity originated from the T7 run-off transcription ( ).

    Article Title: An mRNA-protein Fusion at N-terminus for Evolutionary Protein Engineering
    Article Snippet: .. Ligation between the mRNA and the hydrazide-linker The mRNA (2 µM) was hybridized to the DNA moiety of the hydrazide-linker (4 µM) and prRT- (4 µM) by heating at 95 °C and cooling to 25 °C in 50 µl of T4 RNA ligase buffer (Takara Bio) and ligation reaction was started by adding T4 RNA ligase (40 U, Takara Bio) and ribonuclease inhibitor (40 U, Takara Bio). .. Ligation reaction was performed at 25 °C for overnight and the ligated product was analyzed by 8 M urea 8 % PAGE using TBE running buffer at 65 °C and were visualized with fluorescence of FITC and then visualized again after staining by SYBR Green II (Cambrex) using a fluorescence imager (Pharos FX; Bio-Rad).

    Article Title: Easy and Rapid Binding Assay for Functional Analysis of Disulfide-Containing Peptides by a Pull-Down Method Using a Puromycin-Linker and a Cell-Free Translation System
    Article Snippet: .. The puromycin-linker was hybridized to the purified mRNA, and the 5'-terminus of the puromycin-linker and the 3'-terminus of the mRNA were ligated with T4 RNA ligase (Takara Bio, Otsu, Japan) and polynucleotide kinase (PNK; Takara Bio) at 25 °C for 1 h. Six picomoles of the ligation product were placed in 50 µL of a cell-free translation reaction solution with the Retic Lysate IVT kit (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 30 °C for 30 min. Then, 20 µL of 3 M KCl and 6 µL of 1 M MgCl2 were added to the reaction solution and incubated at 37 °C for 1 h. Eighteen microliters of EDTA solution (0.5 M, pH 8.0) were added to the translation reaction and incubated at 25 °C for 10 min to remove bound ribosomes. .. The mRNA-linker-peptide fusion molecules were isolated from the translation reaction solution using 30 μL of Dynabeads MyOne Streptavidin C1 (SA-beads; Thermo Fisher Scientific) according to the supplier’s instructions.

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
    Article Snippet: Protocols Hybridize the mRNA (0.5 µM) with Linker-Y (0.6µM) in an annealing condition of 15 min / from-94-to-25 ºC in ligation buffers. .. Ligate the mRNA with the hybridized Linker-Y using T4 RNA ligase (1 U/µl, TAKARA) in the supplier's buffer with 10% DMSO for 15 min at 25 ºC.

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
    Article Snippet: .. We tested another method against mRNA terminal heterogeneity, which was splint ligation with T4 RNA ligase. .. T4 RNA ligase can ligate nucleotides even if the 3' end of mRNA is not a complementary double strand.

    Article Title: The expression profile of microRNAs in mouse embryos
    Article Snippet: .. Ligation was carried out using T4 RNA ligase (Takara Bio Inc.) at 15°C for 15 h. RNA fraction with attached adapters was purified using PAGE gel, phosphorylated by T4 polynucleotide kinase (Takara Bio Inc.) and followed by second round of ligation with the DNA–RNA chimeric 5′-adaptor containing the GAUC site (5′-CCATGTTCGCATCGGCaggauc-3′, RNA is shown in lowercase). .. The ligated product was converted to cDNA by reverse transcriptase (M-MLV RTase, Takara Bio Inc.) with the following primer, 5′-TAATACGACTCACTATAGGG-3′.

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
    Article Snippet: .. The ligation reaction with T4 RNA ligase (1 U/µl, TAKARA) was performed in the supplier's buffer with 10% DMSO at 25 ºC. .. The ligation products were analyzed by 8 M urea 10% PAGE using TBE running buffer at 65 ºC and were visualized with fluorescence of FITC and then visualized again after staining by SYBR Green II (FMC) using an fluorescence imager (Molecular Imager FX; BIO-RAD).

    Article Title: Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling
    Article Snippet: .. Ligation of mRNA to the linker mRNA was annealed to the biotinylated puromycin-linker DNA (1:1 ratio) via the Y-tag sequence in 1× ligase buffer (Takara, Kyoto, Japan) by heating at 94°C and cooling slowly. mRNA and linker were ligated by the addition of T4 kinase (3 U) and T4 RNA ligase (20 U) (Takara) at 25°C for 1 h, then the conjugated product was purified using an RNeasy Kit (Qiagen). .. Ligation efficiency and the purity of the products were checked by polyacrylamide gel electrophoresis using FITC and/or VistraGreen (Molecular Probes, USA) staining on a fluoroimager (Bio-Rad, Hercules, CA, USA).

    Infection:

    Article Title: Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells
    Article Snippet: Human cells were infected with VSV and lysed 16 hr later. .. The lysates were then incubated with T4 RNA ligase (TAKARA) overnight at 37°C to induce intra-molecular DNA joining and produce circular VSV DNA ( ).

    other:

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
    Article Snippet: T4 RNA ligase can ligate nucleotides even if the 3' end of mRNA is not a complementary double strand.

    Polymerase Chain Reaction:

    Article Title: In vitro selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in an Escherichia coli cell-free translation system
    Article Snippet: .. T4 RNA ligase, Bca BEST RNA PCR kit ver1.1, GelStar Nucleic Acid Stain and ribonuclease inhibitor were from TaKaRa BIO. .. Escherichia coli S30 Extract System for Linear Templates and pGEM-T vector were from Promega.

    Article Title: Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells
    Article Snippet: The lysates were then incubated with T4 RNA ligase (TAKARA) overnight at 37°C to induce intra-molecular DNA joining and produce circular VSV DNA ( ). .. The ligated DNA was purified using phenol/chloroform and subjected to PCR with the N1173-F/N681-R primer pair.

    Article Title: The expression profile of microRNAs in mouse embryos
    Article Snippet: Ligation was carried out using T4 RNA ligase (Takara Bio Inc.) at 15°C for 15 h. RNA fraction with attached adapters was purified using PAGE gel, phosphorylated by T4 polynucleotide kinase (Takara Bio Inc.) and followed by second round of ligation with the DNA–RNA chimeric 5′-adaptor containing the GAUC site (5′-CCATGTTCGCATCGGCaggauc-3′, RNA is shown in lowercase). .. The cDNA was amplified by 12 cycles of PCR using Pyrobest DNA Polymerase (Takara Bio Inc.) and PCR primers (5′-CCATGTTCGCATCGGCAGGATC-3′, 5′-AGCCGTCAGCATCATTGAGCAT-3′) in the presence of 5-methylated-dCTP, dATP, dGTP, dTTP mixture.

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
    Article Snippet: The PCR primers and the mRNA sequence are the same as described in ( ). .. The ligation reaction with T4 RNA ligase (1 U/µl, TAKARA) was performed in the supplier's buffer with 10% DMSO at 25 ºC.

    Fluorescence:

    Article Title: An mRNA-protein Fusion at N-terminus for Evolutionary Protein Engineering
    Article Snippet: Ligation between the mRNA and the hydrazide-linker The mRNA (2 µM) was hybridized to the DNA moiety of the hydrazide-linker (4 µM) and prRT- (4 µM) by heating at 95 °C and cooling to 25 °C in 50 µl of T4 RNA ligase buffer (Takara Bio) and ligation reaction was started by adding T4 RNA ligase (40 U, Takara Bio) and ribonuclease inhibitor (40 U, Takara Bio). .. Ligation reaction was performed at 25 °C for overnight and the ligated product was analyzed by 8 M urea 8 % PAGE using TBE running buffer at 65 °C and were visualized with fluorescence of FITC and then visualized again after staining by SYBR Green II (Cambrex) using a fluorescence imager (Pharos FX; Bio-Rad).

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
    Article Snippet: Ligate the mRNA with the hybridized Linker-Y using T4 RNA ligase (1 U/µl, TAKARA) in the supplier's buffer with 10% DMSO for 15 min at 25 ºC. .. Check the FITC fluorescent gel bands using a fluorescence imager (Molecular Imager FX; BIO-RAD), and then check again after staining by SYBR Green II (FMC).

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
    Article Snippet: The ligation reaction with T4 RNA ligase (1 U/µl, TAKARA) was performed in the supplier's buffer with 10% DMSO at 25 ºC. .. The ligation products were analyzed by 8 M urea 10% PAGE using TBE running buffer at 65 ºC and were visualized with fluorescence of FITC and then visualized again after staining by SYBR Green II (FMC) using an fluorescence imager (Molecular Imager FX; BIO-RAD).

    Isolation:

    Article Title: Easy and Rapid Binding Assay for Functional Analysis of Disulfide-Containing Peptides by a Pull-Down Method Using a Puromycin-Linker and a Cell-Free Translation System
    Article Snippet: The puromycin-linker was hybridized to the purified mRNA, and the 5'-terminus of the puromycin-linker and the 3'-terminus of the mRNA were ligated with T4 RNA ligase (Takara Bio, Otsu, Japan) and polynucleotide kinase (PNK; Takara Bio) at 25 °C for 1 h. Six picomoles of the ligation product were placed in 50 µL of a cell-free translation reaction solution with the Retic Lysate IVT kit (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 30 °C for 30 min. Then, 20 µL of 3 M KCl and 6 µL of 1 M MgCl2 were added to the reaction solution and incubated at 37 °C for 1 h. Eighteen microliters of EDTA solution (0.5 M, pH 8.0) were added to the translation reaction and incubated at 25 °C for 10 min to remove bound ribosomes. .. The mRNA-linker-peptide fusion molecules were isolated from the translation reaction solution using 30 μL of Dynabeads MyOne Streptavidin C1 (SA-beads; Thermo Fisher Scientific) according to the supplier’s instructions.

    Article Title: The expression profile of microRNAs in mouse embryos
    Article Snippet: Total RNA was isolated from BALB/c whole embryos (E9.5, E10.5 and E11.5) using Trizol Reagent (Invitrogen). .. Ligation was carried out using T4 RNA ligase (Takara Bio Inc.) at 15°C for 15 h. RNA fraction with attached adapters was purified using PAGE gel, phosphorylated by T4 polynucleotide kinase (Takara Bio Inc.) and followed by second round of ligation with the DNA–RNA chimeric 5′-adaptor containing the GAUC site (5′-CCATGTTCGCATCGGCaggauc-3′, RNA is shown in lowercase).

    Labeling:

    Article Title: In vitro selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in an Escherichia coli cell-free translation system
    Article Snippet: 5′-Phospho-deoxycytidyl-phospho-puromycin (pdCp-puromycin) and a fluorescently labeled primer BFL-YF515 were from Japan Bio Services. .. T4 RNA ligase, Bca BEST RNA PCR kit ver1.1, GelStar Nucleic Acid Stain and ribonuclease inhibitor were from TaKaRa BIO.

    Purification:

    Article Title: An mRNA-protein Fusion at N-terminus for Evolutionary Protein Engineering
    Article Snippet: Ligation between the mRNA and the hydrazide-linker The mRNA (2 µM) was hybridized to the DNA moiety of the hydrazide-linker (4 µM) and prRT- (4 µM) by heating at 95 °C and cooling to 25 °C in 50 µl of T4 RNA ligase buffer (Takara Bio) and ligation reaction was started by adding T4 RNA ligase (40 U, Takara Bio) and ribonuclease inhibitor (40 U, Takara Bio). .. Ligated product was purified using RNeasy Mini Kit (Qiagen).

    Article Title: Easy and Rapid Binding Assay for Functional Analysis of Disulfide-Containing Peptides by a Pull-Down Method Using a Puromycin-Linker and a Cell-Free Translation System
    Article Snippet: .. The puromycin-linker was hybridized to the purified mRNA, and the 5'-terminus of the puromycin-linker and the 3'-terminus of the mRNA were ligated with T4 RNA ligase (Takara Bio, Otsu, Japan) and polynucleotide kinase (PNK; Takara Bio) at 25 °C for 1 h. Six picomoles of the ligation product were placed in 50 µL of a cell-free translation reaction solution with the Retic Lysate IVT kit (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 30 °C for 30 min. Then, 20 µL of 3 M KCl and 6 µL of 1 M MgCl2 were added to the reaction solution and incubated at 37 °C for 1 h. Eighteen microliters of EDTA solution (0.5 M, pH 8.0) were added to the translation reaction and incubated at 25 °C for 10 min to remove bound ribosomes. .. The mRNA-linker-peptide fusion molecules were isolated from the translation reaction solution using 30 μL of Dynabeads MyOne Streptavidin C1 (SA-beads; Thermo Fisher Scientific) according to the supplier’s instructions.

    Article Title: In vitro selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in an Escherichia coli cell-free translation system
    Article Snippet: QIAquick PCR Purification kit and MinElute PCR Purification kit were purchased from QIAGEN. .. T4 RNA ligase, Bca BEST RNA PCR kit ver1.1, GelStar Nucleic Acid Stain and ribonuclease inhibitor were from TaKaRa BIO.

    Article Title: Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells
    Article Snippet: The lysates were then incubated with T4 RNA ligase (TAKARA) overnight at 37°C to induce intra-molecular DNA joining and produce circular VSV DNA ( ). .. The ligated DNA was purified using phenol/chloroform and subjected to PCR with the N1173-F/N681-R primer pair.

    Article Title: The expression profile of microRNAs in mouse embryos
    Article Snippet: .. Ligation was carried out using T4 RNA ligase (Takara Bio Inc.) at 15°C for 15 h. RNA fraction with attached adapters was purified using PAGE gel, phosphorylated by T4 polynucleotide kinase (Takara Bio Inc.) and followed by second round of ligation with the DNA–RNA chimeric 5′-adaptor containing the GAUC site (5′-CCATGTTCGCATCGGCaggauc-3′, RNA is shown in lowercase). .. The ligated product was converted to cDNA by reverse transcriptase (M-MLV RTase, Takara Bio Inc.) with the following primer, 5′-TAATACGACTCACTATAGGG-3′.

    Article Title: Efficacy of a Novel Class of RNA Interference Therapeutic Agents
    Article Snippet: The RNA 5′ end was phosphorylated by treating with T4 polynucleotide kinase (TaKaRa) in a reaction buffer containing 50 pmol RNA, enzyme 20 Units, 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2 , 5 mM DTT and 1 mM ATP at 37°C for 2 h. After phenol/chloroform extraction and ethanol precipitation, the RNA 3′ and 5′ ends were ligated and the RNA converted to a circular type by treating overnight with T4 RNA ligase (TaKaRa) at 16°C in a reaction buffer containing RNA 2 ug, Enzyme 50 U, 50 mM Tris-HCl, pH7.5, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP, 0.006% BSA. .. Dumbbell RNA was cut from the gel, extracted and purified using the small RNA Gel Extraction Kit (TaKaRa) following the manufacturer's instructions.

    Article Title: Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling
    Article Snippet: .. Ligation of mRNA to the linker mRNA was annealed to the biotinylated puromycin-linker DNA (1:1 ratio) via the Y-tag sequence in 1× ligase buffer (Takara, Kyoto, Japan) by heating at 94°C and cooling slowly. mRNA and linker were ligated by the addition of T4 kinase (3 U) and T4 RNA ligase (20 U) (Takara) at 25°C for 1 h, then the conjugated product was purified using an RNeasy Kit (Qiagen). .. Ligation efficiency and the purity of the products were checked by polyacrylamide gel electrophoresis using FITC and/or VistraGreen (Molecular Probes, USA) staining on a fluoroimager (Bio-Rad, Hercules, CA, USA).

    Sequencing:

    Article Title: Easy and Rapid Binding Assay for Functional Analysis of Disulfide-Containing Peptides by a Pull-Down Method Using a Puromycin-Linker and a Cell-Free Translation System
    Article Snippet: The bait peptide-coding DNA template comprised of a T7 promoter, Omega sequence, Kozak sequence, bait-peptide coding region, hexa-histidine-tag, and hybridizing region (HR) of the puromycin-linker ( ). .. The puromycin-linker was hybridized to the purified mRNA, and the 5'-terminus of the puromycin-linker and the 3'-terminus of the mRNA were ligated with T4 RNA ligase (Takara Bio, Otsu, Japan) and polynucleotide kinase (PNK; Takara Bio) at 25 °C for 1 h. Six picomoles of the ligation product were placed in 50 µL of a cell-free translation reaction solution with the Retic Lysate IVT kit (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 30 °C for 30 min. Then, 20 µL of 3 M KCl and 6 µL of 1 M MgCl2 were added to the reaction solution and incubated at 37 °C for 1 h. Eighteen microliters of EDTA solution (0.5 M, pH 8.0) were added to the translation reaction and incubated at 25 °C for 10 min to remove bound ribosomes.

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
    Article Snippet: The PCR primers and the mRNA sequence are the same as described in ( ). .. The ligation reaction with T4 RNA ligase (1 U/µl, TAKARA) was performed in the supplier's buffer with 10% DMSO at 25 ºC.

    Article Title: Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling
    Article Snippet: .. Ligation of mRNA to the linker mRNA was annealed to the biotinylated puromycin-linker DNA (1:1 ratio) via the Y-tag sequence in 1× ligase buffer (Takara, Kyoto, Japan) by heating at 94°C and cooling slowly. mRNA and linker were ligated by the addition of T4 kinase (3 U) and T4 RNA ligase (20 U) (Takara) at 25°C for 1 h, then the conjugated product was purified using an RNeasy Kit (Qiagen). .. Ligation efficiency and the purity of the products were checked by polyacrylamide gel electrophoresis using FITC and/or VistraGreen (Molecular Probes, USA) staining on a fluoroimager (Bio-Rad, Hercules, CA, USA).

    Polyacrylamide Gel Electrophoresis:

    Article Title: An mRNA-protein Fusion at N-terminus for Evolutionary Protein Engineering
    Article Snippet: Ligation between the mRNA and the hydrazide-linker The mRNA (2 µM) was hybridized to the DNA moiety of the hydrazide-linker (4 µM) and prRT- (4 µM) by heating at 95 °C and cooling to 25 °C in 50 µl of T4 RNA ligase buffer (Takara Bio) and ligation reaction was started by adding T4 RNA ligase (40 U, Takara Bio) and ribonuclease inhibitor (40 U, Takara Bio). .. Ligation reaction was performed at 25 °C for overnight and the ligated product was analyzed by 8 M urea 8 % PAGE using TBE running buffer at 65 °C and were visualized with fluorescence of FITC and then visualized again after staining by SYBR Green II (Cambrex) using a fluorescence imager (Pharos FX; Bio-Rad).

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
    Article Snippet: Ligate the mRNA with the hybridized Linker-Y using T4 RNA ligase (1 U/µl, TAKARA) in the supplier's buffer with 10% DMSO for 15 min at 25 ºC. .. Analysis of the ligation products by 8 M urea 10% PAGE using TBE running buffer at 65 ºC.

    Article Title: The expression profile of microRNAs in mouse embryos
    Article Snippet: .. Ligation was carried out using T4 RNA ligase (Takara Bio Inc.) at 15°C for 15 h. RNA fraction with attached adapters was purified using PAGE gel, phosphorylated by T4 polynucleotide kinase (Takara Bio Inc.) and followed by second round of ligation with the DNA–RNA chimeric 5′-adaptor containing the GAUC site (5′-CCATGTTCGCATCGGCaggauc-3′, RNA is shown in lowercase). .. The ligated product was converted to cDNA by reverse transcriptase (M-MLV RTase, Takara Bio Inc.) with the following primer, 5′-TAATACGACTCACTATAGGG-3′.

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
    Article Snippet: The ligation reaction with T4 RNA ligase (1 U/µl, TAKARA) was performed in the supplier's buffer with 10% DMSO at 25 ºC. .. The ligation products were analyzed by 8 M urea 10% PAGE using TBE running buffer at 65 ºC and were visualized with fluorescence of FITC and then visualized again after staining by SYBR Green II (FMC) using an fluorescence imager (Molecular Imager FX; BIO-RAD).

    Article Title: Efficacy of a Novel Class of RNA Interference Therapeutic Agents
    Article Snippet: The RNA 5′ end was phosphorylated by treating with T4 polynucleotide kinase (TaKaRa) in a reaction buffer containing 50 pmol RNA, enzyme 20 Units, 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2 , 5 mM DTT and 1 mM ATP at 37°C for 2 h. After phenol/chloroform extraction and ethanol precipitation, the RNA 3′ and 5′ ends were ligated and the RNA converted to a circular type by treating overnight with T4 RNA ligase (TaKaRa) at 16°C in a reaction buffer containing RNA 2 ug, Enzyme 50 U, 50 mM Tris-HCl, pH7.5, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP, 0.006% BSA. .. After phenol/chloroform extraction and ethanol precipitation, the formation of RNA was confirmed in 7 M urea-denatured 15% PAGE and by ESI-Q-TOF mass spectrometry.

    Article Title: Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling
    Article Snippet: Ligation of mRNA to the linker mRNA was annealed to the biotinylated puromycin-linker DNA (1:1 ratio) via the Y-tag sequence in 1× ligase buffer (Takara, Kyoto, Japan) by heating at 94°C and cooling slowly. mRNA and linker were ligated by the addition of T4 kinase (3 U) and T4 RNA ligase (20 U) (Takara) at 25°C for 1 h, then the conjugated product was purified using an RNeasy Kit (Qiagen). .. Ligation efficiency and the purity of the products were checked by polyacrylamide gel electrophoresis using FITC and/or VistraGreen (Molecular Probes, USA) staining on a fluoroimager (Bio-Rad, Hercules, CA, USA).

    Staining:

    Article Title: An mRNA-protein Fusion at N-terminus for Evolutionary Protein Engineering
    Article Snippet: Ligation between the mRNA and the hydrazide-linker The mRNA (2 µM) was hybridized to the DNA moiety of the hydrazide-linker (4 µM) and prRT- (4 µM) by heating at 95 °C and cooling to 25 °C in 50 µl of T4 RNA ligase buffer (Takara Bio) and ligation reaction was started by adding T4 RNA ligase (40 U, Takara Bio) and ribonuclease inhibitor (40 U, Takara Bio). .. Ligation reaction was performed at 25 °C for overnight and the ligated product was analyzed by 8 M urea 8 % PAGE using TBE running buffer at 65 °C and were visualized with fluorescence of FITC and then visualized again after staining by SYBR Green II (Cambrex) using a fluorescence imager (Pharos FX; Bio-Rad).

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
    Article Snippet: Ligate the mRNA with the hybridized Linker-Y using T4 RNA ligase (1 U/µl, TAKARA) in the supplier's buffer with 10% DMSO for 15 min at 25 ºC. .. Check the FITC fluorescent gel bands using a fluorescence imager (Molecular Imager FX; BIO-RAD), and then check again after staining by SYBR Green II (FMC).

    Article Title: In vitro selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in an Escherichia coli cell-free translation system
    Article Snippet: .. T4 RNA ligase, Bca BEST RNA PCR kit ver1.1, GelStar Nucleic Acid Stain and ribonuclease inhibitor were from TaKaRa BIO. .. Escherichia coli S30 Extract System for Linear Templates and pGEM-T vector were from Promega.

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
    Article Snippet: The ligation reaction with T4 RNA ligase (1 U/µl, TAKARA) was performed in the supplier's buffer with 10% DMSO at 25 ºC. .. The ligation products were analyzed by 8 M urea 10% PAGE using TBE running buffer at 65 ºC and were visualized with fluorescence of FITC and then visualized again after staining by SYBR Green II (FMC) using an fluorescence imager (Molecular Imager FX; BIO-RAD).

    Article Title: Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling
    Article Snippet: Ligation of mRNA to the linker mRNA was annealed to the biotinylated puromycin-linker DNA (1:1 ratio) via the Y-tag sequence in 1× ligase buffer (Takara, Kyoto, Japan) by heating at 94°C and cooling slowly. mRNA and linker were ligated by the addition of T4 kinase (3 U) and T4 RNA ligase (20 U) (Takara) at 25°C for 1 h, then the conjugated product was purified using an RNeasy Kit (Qiagen). .. Ligation efficiency and the purity of the products were checked by polyacrylamide gel electrophoresis using FITC and/or VistraGreen (Molecular Probes, USA) staining on a fluoroimager (Bio-Rad, Hercules, CA, USA).

    Nested PCR:

    Article Title: Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells
    Article Snippet: The lysates were then incubated with T4 RNA ligase (TAKARA) overnight at 37°C to induce intra-molecular DNA joining and produce circular VSV DNA ( ). .. Nested PCR was performed using the N1228-F/N163-R primer pair, and the nested PCR products were cloned into the pGEM®-T Easy vector and sequenced using the SP6 and T7 promoter primers.

    Plasmid Preparation:

    Article Title: In vitro selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in an Escherichia coli cell-free translation system
    Article Snippet: T4 RNA ligase, Bca BEST RNA PCR kit ver1.1, GelStar Nucleic Acid Stain and ribonuclease inhibitor were from TaKaRa BIO. .. Escherichia coli S30 Extract System for Linear Templates and pGEM-T vector were from Promega.

    Article Title: Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells
    Article Snippet: The lysates were then incubated with T4 RNA ligase (TAKARA) overnight at 37°C to induce intra-molecular DNA joining and produce circular VSV DNA ( ). .. Nested PCR was performed using the N1228-F/N163-R primer pair, and the nested PCR products were cloned into the pGEM®-T Easy vector and sequenced using the SP6 and T7 promoter primers.

    Article Title: The expression profile of microRNAs in mouse embryos
    Article Snippet: Ligation was carried out using T4 RNA ligase (Takara Bio Inc.) at 15°C for 15 h. RNA fraction with attached adapters was purified using PAGE gel, phosphorylated by T4 polynucleotide kinase (Takara Bio Inc.) and followed by second round of ligation with the DNA–RNA chimeric 5′-adaptor containing the GAUC site (5′-CCATGTTCGCATCGGCaggauc-3′, RNA is shown in lowercase). .. PCR products were purified, digested by SfaNI (NEB) and cloned into the Tag vector pMBS I (Solexa), linearized with BamHI and BbsI.

    Ethanol Precipitation:

    Article Title: Efficacy of a Novel Class of RNA Interference Therapeutic Agents
    Article Snippet: .. The RNA 5′ end was phosphorylated by treating with T4 polynucleotide kinase (TaKaRa) in a reaction buffer containing 50 pmol RNA, enzyme 20 Units, 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2 , 5 mM DTT and 1 mM ATP at 37°C for 2 h. After phenol/chloroform extraction and ethanol precipitation, the RNA 3′ and 5′ ends were ligated and the RNA converted to a circular type by treating overnight with T4 RNA ligase (TaKaRa) at 16°C in a reaction buffer containing RNA 2 ug, Enzyme 50 U, 50 mM Tris-HCl, pH7.5, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP, 0.006% BSA. .. After phenol/chloroform extraction and ethanol precipitation, the formation of RNA was confirmed in 7 M urea-denatured 15% PAGE and by ESI-Q-TOF mass spectrometry.

    Immunoprecipitation:

    Article Title: A central role for PI3K-AKT signaling pathway in linking SAMHD1-deficiency to the type I interferon signature
    Article Snippet: .. The chemical reagents and antibodies used in this study were purchased from the following manufacturers: poly (I:C) and poly (dA:dT), Sigma; T4 Polynucleotide Kinase and T4 RNA ligase, Takara; T4 RNA Ligase 2 (truncated K227Q) and Antarctic Phosphatase, New England Biolabs (NEB); wortmannin and rapamycin, Sigma; MK-2206, A-674563, and Lapatinib, Selleckchem; Ruxolitinib, Invivogen; mouse monoclonal antibodies to IFNAR1 (Millipore) and SAMHD1 (OriGene); rabbit monoclonal antibodies to IRF3 pho-S386 (Abcam), RIG-I, STING, MyD88, TBK1, TBK1 pho-S172, Stat1 pho-Y701, and GSK-3β (all from Cell Signaling Technology); rabbit polyclonal antibodies to IRF3 (Santa Cruz), SAMHD1 (for immunoprecipitation, Bethyl Lab), GAPDH (Ab Frontier), MAVS, TRIF, IRF7, STAT1, Akt, Akt pho-S473, and GSK-3β pho-S9 (all from Cell Signaling Technology); goat polyclonal antibody to hIL-10Rb (R & D systems). .. All siRNAs were purchased from Dharmacon as ON-Target plus.

    Gel Extraction:

    Article Title: Efficacy of a Novel Class of RNA Interference Therapeutic Agents
    Article Snippet: The RNA 5′ end was phosphorylated by treating with T4 polynucleotide kinase (TaKaRa) in a reaction buffer containing 50 pmol RNA, enzyme 20 Units, 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2 , 5 mM DTT and 1 mM ATP at 37°C for 2 h. After phenol/chloroform extraction and ethanol precipitation, the RNA 3′ and 5′ ends were ligated and the RNA converted to a circular type by treating overnight with T4 RNA ligase (TaKaRa) at 16°C in a reaction buffer containing RNA 2 ug, Enzyme 50 U, 50 mM Tris-HCl, pH7.5, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP, 0.006% BSA. .. Dumbbell RNA was cut from the gel, extracted and purified using the small RNA Gel Extraction Kit (TaKaRa) following the manufacturer's instructions.

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    TaKaRa t4 rna ligase buffer
    Gel electrophoresis pattern of mRNA-linker ligation. The ligation products reacted with or without prRT- DNA oligomer used as a blocker of the 3'-end of mRNA were electrophoresis on 8 M urea 8 % PAGE at 65 °C and were visualized with fluorescence of (A) SYBR Green II and (B) FITC. Lane M: DNA ladder, Lane Y: ligation product, Lane L-: negative control, reaction product without DNA-linker, Lane E-: negative control, reaction product without <t>T4</t> RNA ligase. Mobility of the mRNA-linker and the self-ligation product of mRNA are shown to be equivalent.
    T4 Rna Ligase Buffer, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Gel electrophoresis pattern of mRNA-linker ligation. The ligation products reacted with or without prRT- DNA oligomer used as a blocker of the 3'-end of mRNA were electrophoresis on 8 M urea 8 % PAGE at 65 °C and were visualized with fluorescence of (A) SYBR Green II and (B) FITC. Lane M: DNA ladder, Lane Y: ligation product, Lane L-: negative control, reaction product without DNA-linker, Lane E-: negative control, reaction product without T4 RNA ligase. Mobility of the mRNA-linker and the self-ligation product of mRNA are shown to be equivalent.

    Journal: International Journal of Biological Sciences

    Article Title: An mRNA-protein Fusion at N-terminus for Evolutionary Protein Engineering

    doi:

    Figure Lengend Snippet: Gel electrophoresis pattern of mRNA-linker ligation. The ligation products reacted with or without prRT- DNA oligomer used as a blocker of the 3'-end of mRNA were electrophoresis on 8 M urea 8 % PAGE at 65 °C and were visualized with fluorescence of (A) SYBR Green II and (B) FITC. Lane M: DNA ladder, Lane Y: ligation product, Lane L-: negative control, reaction product without DNA-linker, Lane E-: negative control, reaction product without T4 RNA ligase. Mobility of the mRNA-linker and the self-ligation product of mRNA are shown to be equivalent.

    Article Snippet: Ligation between the mRNA and the hydrazide-linker The mRNA (2 µM) was hybridized to the DNA moiety of the hydrazide-linker (4 µM) and prRT- (4 µM) by heating at 95 °C and cooling to 25 °C in 50 µl of T4 RNA ligase buffer (Takara Bio) and ligation reaction was started by adding T4 RNA ligase (40 U, Takara Bio) and ribonuclease inhibitor (40 U, Takara Bio).

    Techniques: Nucleic Acid Electrophoresis, Ligation, Electrophoresis, Polyacrylamide Gel Electrophoresis, Fluorescence, SYBR Green Assay, Negative Control

    Screening cycle of the mRNA-protein fusion in this study. The dsDNA library is transcribed to mRNA. The mRNA is hybridized to the DNA moiety of the linker having hydrazide group and ligated with T4 RNA ligase. Hydrazide group of the ligated product and acetyl group of the phenylalanine derivative that is acylated to sup tRNA are ligated chemically and the modified mRNA is translated. The modified aminoacyl sup tRNA tends to occupy the A-site of ribosome at UAG codon inserted near downstream of initiation codon and the phenylalanine is incorporated into the growing peptide. Thus, linkage between N-terminus of the nascent peptide and 5'-terminus of its mRNA is achieved. Screening of mRNA-peptide fusion library according to property of the displayed peptide and amplify the genotype molecules of the screened fusions by RT-PCR.

    Journal: International Journal of Biological Sciences

    Article Title: An mRNA-protein Fusion at N-terminus for Evolutionary Protein Engineering

    doi:

    Figure Lengend Snippet: Screening cycle of the mRNA-protein fusion in this study. The dsDNA library is transcribed to mRNA. The mRNA is hybridized to the DNA moiety of the linker having hydrazide group and ligated with T4 RNA ligase. Hydrazide group of the ligated product and acetyl group of the phenylalanine derivative that is acylated to sup tRNA are ligated chemically and the modified mRNA is translated. The modified aminoacyl sup tRNA tends to occupy the A-site of ribosome at UAG codon inserted near downstream of initiation codon and the phenylalanine is incorporated into the growing peptide. Thus, linkage between N-terminus of the nascent peptide and 5'-terminus of its mRNA is achieved. Screening of mRNA-peptide fusion library according to property of the displayed peptide and amplify the genotype molecules of the screened fusions by RT-PCR.

    Article Snippet: Ligation between the mRNA and the hydrazide-linker The mRNA (2 µM) was hybridized to the DNA moiety of the hydrazide-linker (4 µM) and prRT- (4 µM) by heating at 95 °C and cooling to 25 °C in 50 µl of T4 RNA ligase buffer (Takara Bio) and ligation reaction was started by adding T4 RNA ligase (40 U, Takara Bio) and ribonuclease inhibitor (40 U, Takara Bio).

    Techniques: Modification, Reverse Transcription Polymerase Chain Reaction