u t4 ligase  (Thermo Fisher)


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    Name:
    T4 DNA Ligase 1 U µL
    Description:
    T4 DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of ATP between double stranded DNAs with 3 hydroxyl and 5 phosphate termini The unique T4 DNA Ligase buffer optimizes ligation which can be performed in 5 minutes Single stranded nucleic acids are not substrates for this enzyme ApplicationsCloning blunt end or cohesive end ligation and adding linkers or adapters to blunt ended DNASourcePurified from E coli lambda lysogen NM989Performance and quality testingEndodeoxyribonuclease 3 and 5 exodeoxyribonuclease assays ligation efficiency testedUnit definitionOne unit catalyzes the exchange of 1 nmol 32P labeled pyrophosphate into ATP in 20 min at 37°C One unit is equal to approximately 300 cohesive end ligation units Unit reaction conditions66 mM Tris HCl pH 7 6 6 6 mM MgCl2 10 mM DTT 66 µM ATP 3 3 µM 32P labeled pyrophosphate and enzyme in 0 1 mL for 20 min at 37°C
    Catalog Number:
    15224017
    Price:
    None
    Applications:
    ChIP-on-Chip|Cloning|RNAi, Epigenetics & Non-Coding RNA Research|Chromatin Biology|Restriction Enzyme Cloning
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher u t4 ligase
    T4 DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of ATP between double stranded DNAs with 3 hydroxyl and 5 phosphate termini The unique T4 DNA Ligase buffer optimizes ligation which can be performed in 5 minutes Single stranded nucleic acids are not substrates for this enzyme ApplicationsCloning blunt end or cohesive end ligation and adding linkers or adapters to blunt ended DNASourcePurified from E coli lambda lysogen NM989Performance and quality testingEndodeoxyribonuclease 3 and 5 exodeoxyribonuclease assays ligation efficiency testedUnit definitionOne unit catalyzes the exchange of 1 nmol 32P labeled pyrophosphate into ATP in 20 min at 37°C One unit is equal to approximately 300 cohesive end ligation units Unit reaction conditions66 mM Tris HCl pH 7 6 6 6 mM MgCl2 10 mM DTT 66 µM ATP 3 3 µM 32P labeled pyrophosphate and enzyme in 0 1 mL for 20 min at 37°C
    https://www.bioz.com/result/u t4 ligase/product/Thermo Fisher
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    u t4 ligase - by Bioz Stars, 2020-05
    95/100 stars

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    Related Articles

    Nucleic Acid Electrophoresis:

    Article Title: Colonic mucosa-associated diffusely adherent afaC+ Escherichia coli expressing lpfA and pks are increased in inflammatory bowel disease and colon cancer
    Article Snippet: .. The complete afa-1 operon (6.8 kb) was isolated by Xba I/Spe I restriction enzyme digest, purified by gel electrophoresis, ligated into pUC18 using T4 DNA-ligase and propagated in E coli One-ShotTOP10 (Invitrogen) selected on 100 µg/mL ampicillin agar. pUCAfa was then used to transform chemically competent E coli EPI300-T1 containing empty fosmid, to allow direct comparison with E coli 8H8. .. The presence and orientation of afa-1 within pUCAfa were confirmed by PCR.

    Chromatography:

    Article Title: New insight into the mechanism of mitochondrial cytochrome c function
    Article Snippet: .. Materials and methods Material used in this studies were: components for the culture media and buffer solutions for chromatography and electrophoresis (AppliChem, Germany), ampicillin, cytochrome c from equine heart (Sigma, United States), Xho I restriction endonuclease (Promega, USA), Bam H I restriction endonuclease (New England Biolabs Inc., USA), Pfu -DNA polymerase, and T4-DNA ligase (Fermentas, Lithuania). .. Distilled water was additionally purified on a Milli-Q system (Millipore, USA).

    Ligation:

    Article Title: Design of a New [PSI+]-No-More Mutation in SUP35 With Strong Inhibitory Effect on the [PSI+] Prion Propagation
    Article Snippet: .. Sticky-end ligation was performed with T4 DNA-ligase according to Thermo Scientific protocol. pRS315CG was obtained analogously from pRS316CG (Serio et al., ) and pRS315. pR16CUP-NM-yTagRFP-T plasmid was obtained by insertion of the XhoI-XhoI fragment from pCUP-NM-His6 (Kiktev et al., ) in place of the XhoI-SalI fragment of pR16CUP-SFP1C-yTagRFP-T which in turn resulted from the substitution of the PstI-PstI fragment in pR16CUP-SFP1-Cerulean (Matveenko et al., ) for the PstI-PstI fragment from pIM35 (Malcova et al., ). ..

    Isolation:

    Article Title: Colonic mucosa-associated diffusely adherent afaC+ Escherichia coli expressing lpfA and pks are increased in inflammatory bowel disease and colon cancer
    Article Snippet: .. The complete afa-1 operon (6.8 kb) was isolated by Xba I/Spe I restriction enzyme digest, purified by gel electrophoresis, ligated into pUC18 using T4 DNA-ligase and propagated in E coli One-ShotTOP10 (Invitrogen) selected on 100 µg/mL ampicillin agar. pUCAfa was then used to transform chemically competent E coli EPI300-T1 containing empty fosmid, to allow direct comparison with E coli 8H8. .. The presence and orientation of afa-1 within pUCAfa were confirmed by PCR.

    Purification:

    Article Title: Colonic mucosa-associated diffusely adherent afaC+ Escherichia coli expressing lpfA and pks are increased in inflammatory bowel disease and colon cancer
    Article Snippet: .. The complete afa-1 operon (6.8 kb) was isolated by Xba I/Spe I restriction enzyme digest, purified by gel electrophoresis, ligated into pUC18 using T4 DNA-ligase and propagated in E coli One-ShotTOP10 (Invitrogen) selected on 100 µg/mL ampicillin agar. pUCAfa was then used to transform chemically competent E coli EPI300-T1 containing empty fosmid, to allow direct comparison with E coli 8H8. .. The presence and orientation of afa-1 within pUCAfa were confirmed by PCR.

    Electrophoresis:

    Article Title: New insight into the mechanism of mitochondrial cytochrome c function
    Article Snippet: .. Materials and methods Material used in this studies were: components for the culture media and buffer solutions for chromatography and electrophoresis (AppliChem, Germany), ampicillin, cytochrome c from equine heart (Sigma, United States), Xho I restriction endonuclease (Promega, USA), Bam H I restriction endonuclease (New England Biolabs Inc., USA), Pfu -DNA polymerase, and T4-DNA ligase (Fermentas, Lithuania). .. Distilled water was additionally purified on a Milli-Q system (Millipore, USA).

    Incubation:

    Article Title: Inhibition of PTTG1 expression by microRNA suppresses proliferation and induces apoptosis of malignant glioma cells
    Article Snippet: .. The oligonucleotides (10 µM) were incubated in Invitrogen annealing buffer (Thermo Fisher Scientific, Inc.) at 95°C for 4 min and 37°C for 10 min prior to being ligated to the empty vector using Invitrogen T4 ligase (Thermo Fisher Scientific, Inc.). .. Recombinant vectors were used to transform competent cells, which were then seeded onto lysogeny broth (LB)-spectinomycin culture plates (BD Biosciences, Franklin Lakes, NJ, USA).

    other:

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
    Article Snippet: T4 DNA ligase (Cat. #15224–017) and T4 DNA polymerase (Cat. #18005–017) were from Invitrogen.

    Transformation Assay:

    Article Title: Nickel-induced HIF-1α promotes growth arrest and senescence in normal human cells but lacks toxic effects in transformed cells
    Article Snippet: .. Oligonucleotides containing targeting sequences and vector-compatible ends were ligated into the linearized pSUPER.retro.puro using T4 ligase (15224017, Invitrogen) overnight followed by the transformation of the plasmid products into One Shot TOPO10 Chemically Competent E. coli cells (C404003, Invitrogen). .. The viral particles were produced in 293T cells by cotransfection of pSUPER DNA with plasmids expressing MoMuLV gag-pol and VSVG.

    Plasmid Preparation:

    Article Title: Nickel-induced HIF-1α promotes growth arrest and senescence in normal human cells but lacks toxic effects in transformed cells
    Article Snippet: .. Oligonucleotides containing targeting sequences and vector-compatible ends were ligated into the linearized pSUPER.retro.puro using T4 ligase (15224017, Invitrogen) overnight followed by the transformation of the plasmid products into One Shot TOPO10 Chemically Competent E. coli cells (C404003, Invitrogen). .. The viral particles were produced in 293T cells by cotransfection of pSUPER DNA with plasmids expressing MoMuLV gag-pol and VSVG.

    Article Title: Inhibition of PTTG1 expression by microRNA suppresses proliferation and induces apoptosis of malignant glioma cells
    Article Snippet: .. The oligonucleotides (10 µM) were incubated in Invitrogen annealing buffer (Thermo Fisher Scientific, Inc.) at 95°C for 4 min and 37°C for 10 min prior to being ligated to the empty vector using Invitrogen T4 ligase (Thermo Fisher Scientific, Inc.). .. Recombinant vectors were used to transform competent cells, which were then seeded onto lysogeny broth (LB)-spectinomycin culture plates (BD Biosciences, Franklin Lakes, NJ, USA).

    Article Title: Design of a New [PSI+]-No-More Mutation in SUP35 With Strong Inhibitory Effect on the [PSI+] Prion Propagation
    Article Snippet: .. Sticky-end ligation was performed with T4 DNA-ligase according to Thermo Scientific protocol. pRS315CG was obtained analogously from pRS316CG (Serio et al., ) and pRS315. pR16CUP-NM-yTagRFP-T plasmid was obtained by insertion of the XhoI-XhoI fragment from pCUP-NM-His6 (Kiktev et al., ) in place of the XhoI-SalI fragment of pR16CUP-SFP1C-yTagRFP-T which in turn resulted from the substitution of the PstI-PstI fragment in pR16CUP-SFP1-Cerulean (Matveenko et al., ) for the PstI-PstI fragment from pIM35 (Malcova et al., ). ..

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  • 99
    Thermo Fisher t4 rna ligase
    Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using <t>T4</t> RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).
    T4 Rna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase/product/Thermo Fisher
    Average 99 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase - by Bioz Stars, 2020-05
    99/100 stars
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    Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using T4 RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).

    Journal: RNA Biology

    Article Title: Identification of host RNAs that interact with EBV noncoding RNA EBER2

    doi: 10.1080/15476286.2018.1518854

    Figure Lengend Snippet: Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using T4 RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).

    Article Snippet: RNA was resuspended in 14.5 µl H2 O and subjected to T4 RNA Ligase reaction by adding 1 µl of 20 µM 5′-phosporylated RL3 (5′-P -GUGUCAGUCACUUCCAGCGG-Puromycin-3′), 2 µl 10× T4 Ligase Buffer, 2 µl BSA, 0.5 µl T4 RNA Ligase (ThermoFisher), and incubated overnight at 16°C.

    Techniques: Allele-specific Oligonucleotide, Selection, In Vivo, Generated, Sequencing, Combined Bisulfite Restriction Analysis Assay, Activity Assay, shRNA, Protein Concentration, Size-exclusion Chromatography, Ligation, Staining, Purification, Sample Prep, Labeling, Autoradiography

    Preparation and analysis on circular RNA in vitro . (A) Schematic of in vitro circularization constructs. Transcripts to be circularized consist of a terminal 10 nt open loop structure (black) and a reverse-complementary repeat sequence of 11 nt, which forms an intramolecular stem (red). This structure is followed by a 63 nt constant region for detection by northern blot or PCR (blue), followed by the miRNA-122 sponge (bulge; perfect) or a scrambled control sequence (shuffle) in grey. (B) Schematic of the in vitro ligation reaction. 4-fold excess of GMP over GTP results in ∼80% of the transcripts containing a 5′-monophosphate, enabling efficient in vitro ligation by T4 RNA ligase. Ligation products are circular RNAs (intramolecular ligation) or linear dimers (intermolecular ligation). (C) In vitro ligation reactions described in (B) were analyzed on 5%, 6% or 7% polyacrylamide-urea gels by ethidium bromide staining. While mobility of linear RNAs remains unchanged compared to RNA marker, the apparent mobility of circular RNA is lower in higher percentage gels (indicated by dash/double dash or circle). (D) Purified linear or circular RNAs from (C) were transfected in HuH-7.5 cells and total RNA was prepared after 4, 8, 14, 24 and 32 h. RNAs were detected by ³²P-northern blot analysis using identical probes in the constant region [labeled blue in (A)]. (E) HuH-7.5 cells transfected with circular RNA or linear RNA from (C) were subjected to sub-cellular fractionation and cytoplasmic or nuclear fractions were analyzed by ³²P-northern blot detecting transfected RNAs along with U1 snRNA and by western blot against hnRNP A1 or GAPDH proteins as a fractionation control. In the circRNA-transfected samples, a degradation product is detected at linear monomer size (“linearized”).

    Journal: RNA Biology

    Article Title: Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges

    doi: 10.1080/15476286.2018.1435248

    Figure Lengend Snippet: Preparation and analysis on circular RNA in vitro . (A) Schematic of in vitro circularization constructs. Transcripts to be circularized consist of a terminal 10 nt open loop structure (black) and a reverse-complementary repeat sequence of 11 nt, which forms an intramolecular stem (red). This structure is followed by a 63 nt constant region for detection by northern blot or PCR (blue), followed by the miRNA-122 sponge (bulge; perfect) or a scrambled control sequence (shuffle) in grey. (B) Schematic of the in vitro ligation reaction. 4-fold excess of GMP over GTP results in ∼80% of the transcripts containing a 5′-monophosphate, enabling efficient in vitro ligation by T4 RNA ligase. Ligation products are circular RNAs (intramolecular ligation) or linear dimers (intermolecular ligation). (C) In vitro ligation reactions described in (B) were analyzed on 5%, 6% or 7% polyacrylamide-urea gels by ethidium bromide staining. While mobility of linear RNAs remains unchanged compared to RNA marker, the apparent mobility of circular RNA is lower in higher percentage gels (indicated by dash/double dash or circle). (D) Purified linear or circular RNAs from (C) were transfected in HuH-7.5 cells and total RNA was prepared after 4, 8, 14, 24 and 32 h. RNAs were detected by ³²P-northern blot analysis using identical probes in the constant region [labeled blue in (A)]. (E) HuH-7.5 cells transfected with circular RNA or linear RNA from (C) were subjected to sub-cellular fractionation and cytoplasmic or nuclear fractions were analyzed by ³²P-northern blot detecting transfected RNAs along with U1 snRNA and by western blot against hnRNP A1 or GAPDH proteins as a fractionation control. In the circRNA-transfected samples, a degradation product is detected at linear monomer size (“linearized”).

    Article Snippet: Next, T4 RNA ligase buffer and RNaseOUT (Thermo Fisher Scientific) were added and incubated for 10 min at 37°C.

    Techniques: In Vitro, Construct, Sequencing, Northern Blot, Polymerase Chain Reaction, Ligation, Staining, Marker, Purification, Transfection, Labeling, Cell Fractionation, Western Blot, Fractionation

    Experimental workflow – cell lysis, miRNA release, capture via 3′ adaptor ligation, followed by 5′ adaptor ligation for PCR amplification and library preparation 3′ adaptor is pre-adenylated at the 3′ end before ligation with miRNA catalyzed by T4 RNA ligase II (w/o ATP). The PCR adaptor coupling is completed via ligation to 5′ adaptor using T4 RNA ligase I (with ATP). This workflow is used to amplify miRNAs and quantify the expression globally at the genome-scale. Moreover, two optional size selection processes were performed using gel purification after each ligation step and before amplification. The two steps in orange highlight the major improvements reported in this study.

    Journal: The Analyst

    Article Title: Capture, Amplification, and Global Profiling of microRNAs from Low Quantities of Whole Cell Lysate

    doi: 10.1039/c7an00670e

    Figure Lengend Snippet: Experimental workflow – cell lysis, miRNA release, capture via 3′ adaptor ligation, followed by 5′ adaptor ligation for PCR amplification and library preparation 3′ adaptor is pre-adenylated at the 3′ end before ligation with miRNA catalyzed by T4 RNA ligase II (w/o ATP). The PCR adaptor coupling is completed via ligation to 5′ adaptor using T4 RNA ligase I (with ATP). This workflow is used to amplify miRNAs and quantify the expression globally at the genome-scale. Moreover, two optional size selection processes were performed using gel purification after each ligation step and before amplification. The two steps in orange highlight the major improvements reported in this study.

    Article Snippet: Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture.

    Techniques: Lysis, Ligation, Polymerase Chain Reaction, Amplification, Expressing, Selection, Gel Purification