t4 ligase buffer  (Thermo Fisher)


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    Structured Review

    Thermo Fisher t4 ligase buffer
    T4 Ligase Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 ligase buffer/product/Thermo Fisher
    Average 94 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    t4 ligase buffer - by Bioz Stars, 2020-05
    94/100 stars

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    Incubation:

    Article Title: Monitoring the T-Cell Receptor Repertoire at Single-Clone Resolution
    Article Snippet: .. 1 pmol of hexamers, 4 units of T4 DNA ligase and 2 µl 5× DNA Ligase buffer (In vitrogen - Life Technologies, Breda, NL) and template/annealer complex were added in a total volume of 10 µl and incubated for 45 minutes at 16°C, followed by a 10 minutes denaturation step at 65°C. .. Ligation products were analyzed on the ABI Prism 3100 Genetic Analyzer capillary system and Genescan software as described above.

    DNA Ligation:

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain
    Article Snippet: .. Ligations of the linkers with 5′-OH ends The ligations of linkers A–B, C–D, and E–F by using T4 DNA ligase were performed in 100 µl of T4 DNA ligase reaction mixture containing 1 x T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, and 0.5 mM ATP; pH 7.8 at 25°C), 1 µM of each oligo, and 0.25 Weiss units/µl of T4 DNA ligase (Fermentas, Lithuania; Promega, USA; and Takara, Japan). .. The ligations of linkers A–B, C–D, and E–F by using E. coli DNA ligase were performed in 50 µl of E. coli DNA ligase reaction mixture containing 1 x E. coli DNA ligation buffer (30 mM Tris-HCl, 4 mM MgCl2 , 10 mM (NH4 )2 SO4 , 1.2 mM EDTA, and 0.1 mM NAD+ ; pH 8.0 at 25°C), 1 x BSA (0.005%), 2 µM of each oligo, and 6 U/µl of E. coli DNA ligase (Takara).

    Article Title: SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing
    Article Snippet: .. For the 5΄-end ligation; ss2 (final conc 10 μM), T4 DNA ligation buffer, PEG4000 (5%, w/v) and 30 units T4 DNA ligase (Thermo Fisher Scientific) and nuclease free H2 O was added to the sample on ice, in a total volume of 20 μl. .. Ligation reaction mixtures were incubated at 20°C for 1 h, purified using AMPureXP beads in a 2:1 bead to sample ratio and eluted in 10 μl nuclease free water.

    Plasmid Preparation:

    Article Title: The COMET toolkit for composing customizable genetic programs in mammalian cells
    Article Snippet: .. BpiI Golden Gate reaction mixtures comprise 1 µL T4 ligase buffer, 1 µL 10× BSA (1 mg/mL), 0.4 µL BpiI-FD (Thermo Fisher), 0.4 µL µL T4 Ligase (400 U/µL; NEB), 10 fmol of vector, 1 µL of each insert (diluted to 200 nM or 20 nM), and water to 10 µL. .. Non-Golden Gate assembly of some ZF reporters Although Golden Gate assembly was the primary strategy for cloning the promoters, the first-generation templates were not readily amenable to synthesis and insertion of ZF binding site arrays.

    Ligation:

    Article Title: Phenotypic and Genotypic Analysis of Newly Obtained Interspecific Hybrids in the Campanula Genus
    Article Snippet: .. The ligation mix containing 1 x T4 DNA ligase-buffer with ATP, 25 μM Mse1 , 2.5 μM Pstl and 2 U/μl T4 DNA ligase (Fermentas, Thermo Scientific, Slangerup, Denmark) was added to the digestion reaction. .. After incubation for 3 hours at 37°C, all samples were diluted 10 fold with ddH2 O. Pre-amplification was performed by adding 1x Extra buffer (15 mM MgCl2 ), 4 mM dNTP-Mix and pre-selective Pstl and Msel primers and 5 U/μl Taq-DNA-polymerase together.

    Article Title: SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing
    Article Snippet: .. For the 5΄-end ligation; ss2 (final conc 10 μM), T4 DNA ligation buffer, PEG4000 (5%, w/v) and 30 units T4 DNA ligase (Thermo Fisher Scientific) and nuclease free H2 O was added to the sample on ice, in a total volume of 20 μl. .. Ligation reaction mixtures were incubated at 20°C for 1 h, purified using AMPureXP beads in a 2:1 bead to sample ratio and eluted in 10 μl nuclease free water.

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    Thermo Fisher t4 rna ligase buffer
    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 <t>T4</t> 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”).
    T4 Rna Ligase Buffer, 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 buffer/product/Thermo Fisher
    Average 99 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase buffer - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    96
    Thermo Fisher t4 dna ligase buffer
    Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard <t>T4</t> DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.
    T4 Dna Ligase Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase buffer/product/Thermo Fisher
    Average 96 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase buffer - by Bioz Stars, 2020-05
    96/100 stars
      Buy from Supplier

    94
    Thermo Fisher t4 dna ligation buffer
    Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard <t>T4</t> DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.
    T4 Dna Ligation Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligation buffer/product/Thermo Fisher
    Average 94 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligation buffer - by Bioz Stars, 2020-05
    94/100 stars
      Buy from Supplier

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    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

    Schematic representation of radiolabeling of RNA at its 3′ end. T4 RNA ligase catalyzes the ligation reaction where 5′[ 32 P]pCp is covalently attached to the 3′ end of the single-stranded RNA substrate. The radiolabeled RNA molecule

    Journal: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]

    Article Title: Synthesis and Labeling of RNA In Vitro

    doi: 10.1002/0471142727.mb0415s102

    Figure Lengend Snippet: Schematic representation of radiolabeling of RNA at its 3′ end. T4 RNA ligase catalyzes the ligation reaction where 5′[ 32 P]pCp is covalently attached to the 3′ end of the single-stranded RNA substrate. The radiolabeled RNA molecule

    Article Snippet: 10 × buffer for T4 RNA ligase (see recipe) 10 mM ATP (Thermo Scientific) RNA substrate with 3′ hydroxyl end derived from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 5′ 10 µCi/µl [32 P]pCp (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 RNA ligase (Thermo Scientific) G50 buffer (see recipe) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for T4 RNA ligase 1 µl distilled, deionized H2 O 1 µl 10 mM ATP 5 µl RNA substrate with a 3′-hydroxyl end (30 pmol) 10 µl 10 µCi/µl 5′ [32 P]pCp (3000 Ci/mmol) 1 µl 10 U/µl T4 RNA ligase.

    Techniques: Radioactivity, Ligation

    Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard T4 DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.

    Journal: Nucleic Acids Research

    Article Title: SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing

    doi: 10.1093/nar/gkw1110

    Figure Lengend Snippet: Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard T4 DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.

    Article Snippet: For the 3΄-end ligation; adapter ss1 (final conc 10 μM), T4 DNA ligase buffer (40 mM Tris–HCl pH 7.8,10 mM MgCl2 , 10 mM DTT, 0.5 mM ATP), PEG4000 (5% w/v) and 30 units T4 DNA ligase (Thermo Fisher Scientific) and nuclease free water was added to the sample on ice, in a total volume of 30 μl.

    Techniques: Bisulfite Sequencing, Methylation, Construct, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing, Sequencing, DNA Methylation Assay, Random Hexamer Labeling, DNA Ligation, Modification, Ligation

    Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard T4 DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.

    Journal: Nucleic Acids Research

    Article Title: SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing

    doi: 10.1093/nar/gkw1110

    Figure Lengend Snippet: Principles of library preparation methods for whole genome bisulphite sequencing. In the conventional workflow (MethylC-seq) methylated adapters are ligated to double stranded sheared DNA fragments. The constructs are then bisulphite converted prior to amplification with a uracil reading PCR polymerase. The Accel-NGS Methyl-Seq uses the proprietary Adaptase™ technology to attach a low complexity sequence tail to the 3΄-termini of pre-sheared and bisulphite-converted DNA, and an adapter sequence. After an extension step a second adapter is ligated and the libraries are PCR amplified. The TruSeq DNA Methylation method (formerly EpiGnome) uses random hexamer tagged oligonucleotides to simultaneously copy the bisulphite-converted strand and add a 5΄-terminal adaptor sequence. In a subsequent step, a 3΄-terminal adapter is tagged, also by using a random sequence oligonucleotide. In the SPLAT protocol adapters with a protruding random hexamer are annealed to the 3΄-termini of the single stranded DNA. The random hexamer acts as a ‘splint’ and the adapter sequence is ligated to the 3΄-termini of single stranded DNA using standard T4 DNA ligation. A modification of the last 3΄- residue of the random hexamer is required to prevent self-ligation of the adapter. In a second step, adapters with a 5΄-terminal random hexamer overhang is annealed to ligate the 5΄-termini of the single stranded DNA, also using T4 DNA ligase. Finally the SPLAT libraries are PCR amplified using a uracil reading polymerase.

    Article Snippet: For the 5΄-end ligation; ss2 (final conc 10 μM), T4 DNA ligation buffer, PEG4000 (5%, w/v) and 30 units T4 DNA ligase (Thermo Fisher Scientific) and nuclease free H2 O was added to the sample on ice, in a total volume of 20 μl.

    Techniques: Bisulfite Sequencing, Methylation, Construct, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing, Sequencing, DNA Methylation Assay, Random Hexamer Labeling, DNA Ligation, Modification, Ligation