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: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 ligase buffer/product/Thermo Fisher
    Average 99 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    t4 ligase buffer - by Bioz Stars, 2020-01
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Reciprocal chromosome translocation associated with TDNA-insertion mutation in Arabidopsis: genetic and cytological analyses of consequences for gametophyte development and for construction of doubly mutant lines
    Article Snippet: Briefly, genomic AtREV3 2/ 2 DNA (200 ng), 0.28 μM “Eco” Adapter DNA, and 0.28 μM “Hind” adapter DNA were digested with 50 U Eco RI endonuclease (New England Biolabs, Ipswich, MA, USA) and 5 U Hin dIII endonuclease (Invitrogen, Carlsbad, CA, USA) in the presence of 5 U T4 DNA-ligase (Invitrogen) for 6 h at 37°C in 1× T4 ligase buffer [40 mM Tris–HCl (pH 7.8 at 25°C), 10 mM MgCl2 , 10 mM dithiothreitol, 0.5 mM ATP (Fermentas, Glen Burnie, MD, USA)]. .. PCR amplification was carried out for 10 cycles at 96°C for 20 s and 72°C for 140 s, followed by 15 cycles at 96°C for 20 s and 67°C for 140 s. PCR reactions were diluted 1:5 in distilled H2 O; 1.5 μl of the diluted product was used as the template for a second (“nested”) PCR reaction using primers LBb1 and AP2 ( ): 5 cycles at 96°C for 30 s, 94°C for 20 s and 72°C for 140 s, followed by 23 cycles at 96°C for 20 s, 67°C for 20 s and 72°C for 130 s. Products from the nested PCR reaction were cloned in vector pCR®4 using a TOPO TA Cloning Kit for Sequencing (Invitrogen) and transformed into chemically competent E. coli DH5α by standard procedures.

    Article Title: Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata
    Article Snippet: Paragraph title: Cloning and sequencing ... The purified PCR product was ligated into pGEM® -T Easy vector (Promega) using a 1:4 M ratio (vector: PCR product) by incubating the mixture with T4-DNA ligase and T4 ligase buffer at room temperature for 3 h. The ligation reaction mix (5 μl) was used to transform 50 μl of TOP10 Escherichia coli competent cells (Invitrogen).

    Amplification:

    Article Title: Reciprocal chromosome translocation associated with TDNA-insertion mutation in Arabidopsis: genetic and cytological analyses of consequences for gametophyte development and for construction of doubly mutant lines
    Article Snippet: Briefly, genomic AtREV3 2/ 2 DNA (200 ng), 0.28 μM “Eco” Adapter DNA, and 0.28 μM “Hind” adapter DNA were digested with 50 U Eco RI endonuclease (New England Biolabs, Ipswich, MA, USA) and 5 U Hin dIII endonuclease (Invitrogen, Carlsbad, CA, USA) in the presence of 5 U T4 DNA-ligase (Invitrogen) for 6 h at 37°C in 1× T4 ligase buffer [40 mM Tris–HCl (pH 7.8 at 25°C), 10 mM MgCl2 , 10 mM dithiothreitol, 0.5 mM ATP (Fermentas, Glen Burnie, MD, USA)]. .. PCR amplification was carried out for 10 cycles at 96°C for 20 s and 72°C for 140 s, followed by 15 cycles at 96°C for 20 s and 67°C for 140 s. PCR reactions were diluted 1:5 in distilled H2 O; 1.5 μl of the diluted product was used as the template for a second (“nested”) PCR reaction using primers LBb1 and AP2 ( ): 5 cycles at 96°C for 30 s, 94°C for 20 s and 72°C for 140 s, followed by 23 cycles at 96°C for 20 s, 67°C for 20 s and 72°C for 130 s. Products from the nested PCR reaction were cloned in vector pCR®4 using a TOPO TA Cloning Kit for Sequencing (Invitrogen) and transformed into chemically competent E. coli DH5α by standard procedures.

    TA Cloning:

    Article Title: Reciprocal chromosome translocation associated with TDNA-insertion mutation in Arabidopsis: genetic and cytological analyses of consequences for gametophyte development and for construction of doubly mutant lines
    Article Snippet: Briefly, genomic AtREV3 2/ 2 DNA (200 ng), 0.28 μM “Eco” Adapter DNA, and 0.28 μM “Hind” adapter DNA were digested with 50 U Eco RI endonuclease (New England Biolabs, Ipswich, MA, USA) and 5 U Hin dIII endonuclease (Invitrogen, Carlsbad, CA, USA) in the presence of 5 U T4 DNA-ligase (Invitrogen) for 6 h at 37°C in 1× T4 ligase buffer [40 mM Tris–HCl (pH 7.8 at 25°C), 10 mM MgCl2 , 10 mM dithiothreitol, 0.5 mM ATP (Fermentas, Glen Burnie, MD, USA)]. .. PCR amplification was carried out for 10 cycles at 96°C for 20 s and 72°C for 140 s, followed by 15 cycles at 96°C for 20 s and 67°C for 140 s. PCR reactions were diluted 1:5 in distilled H2 O; 1.5 μl of the diluted product was used as the template for a second (“nested”) PCR reaction using primers LBb1 and AP2 ( ): 5 cycles at 96°C for 30 s, 94°C for 20 s and 72°C for 140 s, followed by 23 cycles at 96°C for 20 s, 67°C for 20 s and 72°C for 130 s. Products from the nested PCR reaction were cloned in vector pCR®4 using a TOPO TA Cloning Kit for Sequencing (Invitrogen) and transformed into chemically competent E. coli DH5α by standard procedures.

    Construct:

    Article Title: Production of a recombinant membrane protein in an Escherichia coli strain for the whole cell biosynthesis of phenylacetic acids
    Article Snippet: The pEX-A-styC construct was transferred into E. coli DH5α via heat–shock transformation for propagation. .. Afterwards, 6 ng of the styC -containing insert and 16 ng digested pET16bP (molar ratio 4:1) were ligated in 1 × T4 ligase buffer containing 1 U of T4 ligase (Thermo Scientific).

    Incubation:

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins
    Article Snippet: .. Adaptor ligation The treated samples (±Dpn I) were added to 2 µl 10× T4 ligase buffer, 1 µl of 50 µM dsOligos AdRt/AdRb, 2.5 units of T4 DNA ligase (Thermo Fisher Scientific, EL0011) and 6.5 µl H2 O, and incubated overnight at 16-18°C. .. The reaction was cleaned up using an InnuPREP Double EPure Kit (Analytik Jena) and eluted in 50 µl of H2 O.

    Article Title: 3C-digital PCR for quantification of chromatin interactions
    Article Snippet: .. To stop the restriction digestion, 1.6% SDS (final concentration) was added, and samples were incubated at 65 °C for 20 min. Ligation were performed at 16 °C for 4 h in 15 mL tubes containing 745 μL 10× T4 ligase Buffer, 10% Triton-X 100, 80 μL 10 mg/mL BSA, 6 mL water, 575 μL of cell lysate, 10 μL 1U/μL T4 ligase (Invitrogen, Grand Island, NY, USA). .. The crosslinks were reversed with Proteinase K (Invitrogen) at 65 °C overnight.

    Article Title: Solid-phase cloning for high-throughput assembly of single and multiple DNA parts
    Article Snippet: .. Supernatant from the bead release was mixed with 34 μl water, 0.67 μl ATP (10 mM), 4 μl T4 Ligase buffer (10×), 1.33 μl T4 Ligase (Thermo Fischer Scientific Inc, Waltham, MA, USA) and incubated for 45 min at room temperature. .. Transformation Eight microliters from the ligation mixture was mixed with 2 μl of 5× 500 mM KCl, 150 mM CaCl2 , 250 mM MgCl2 (KCM) and incubated on ice for 3 min before 10 μl of TOP10 chemically competent cells were added ( ).

    Article Title: Prostate cancer risk locus at 8q24 as a regulatory hub by physical interactions with multiple genomic loci across the genome
    Article Snippet: .. The restriction endonuclease was inactivated by the addition of SDS to a final concentration of 1.6% and incubated at 65°C for 20 min. Ligation mixes, prepared in 15-mL tubes containing 745 μl of 10× T4 Ligase buffer, 10% Triton-X 100, 80 μl of 10 mg/mL BSA, 6 ml of water, 575 μl of cell lysate and 10 μl of 1 U/μl T4 ligase (Invitrogen, Grand Island, NY), were incubated at 16°C for 4 h and then at room temperature for 30 min. ..

    Transformation Assay:

    Article Title: Production of a recombinant membrane protein in an Escherichia coli strain for the whole cell biosynthesis of phenylacetic acids
    Article Snippet: The pEX-A-styC construct was transferred into E. coli DH5α via heat–shock transformation for propagation. .. Afterwards, 6 ng of the styC -containing insert and 16 ng digested pET16bP (molar ratio 4:1) were ligated in 1 × T4 ligase buffer containing 1 U of T4 ligase (Thermo Scientific).

    Article Title: Reciprocal chromosome translocation associated with TDNA-insertion mutation in Arabidopsis: genetic and cytological analyses of consequences for gametophyte development and for construction of doubly mutant lines
    Article Snippet: Briefly, genomic AtREV3 2/ 2 DNA (200 ng), 0.28 μM “Eco” Adapter DNA, and 0.28 μM “Hind” adapter DNA were digested with 50 U Eco RI endonuclease (New England Biolabs, Ipswich, MA, USA) and 5 U Hin dIII endonuclease (Invitrogen, Carlsbad, CA, USA) in the presence of 5 U T4 DNA-ligase (Invitrogen) for 6 h at 37°C in 1× T4 ligase buffer [40 mM Tris–HCl (pH 7.8 at 25°C), 10 mM MgCl2 , 10 mM dithiothreitol, 0.5 mM ATP (Fermentas, Glen Burnie, MD, USA)]. .. PCR amplification was carried out for 10 cycles at 96°C for 20 s and 72°C for 140 s, followed by 15 cycles at 96°C for 20 s and 67°C for 140 s. PCR reactions were diluted 1:5 in distilled H2 O; 1.5 μl of the diluted product was used as the template for a second (“nested”) PCR reaction using primers LBb1 and AP2 ( ): 5 cycles at 96°C for 30 s, 94°C for 20 s and 72°C for 140 s, followed by 23 cycles at 96°C for 20 s, 67°C for 20 s and 72°C for 130 s. Products from the nested PCR reaction were cloned in vector pCR®4 using a TOPO TA Cloning Kit for Sequencing (Invitrogen) and transformed into chemically competent E. coli DH5α by standard procedures.

    Derivative Assay:

    Article Title: Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata
    Article Snippet: The purified PCR product was ligated into pGEM® -T Easy vector (Promega) using a 1:4 M ratio (vector: PCR product) by incubating the mixture with T4-DNA ligase and T4 ligase buffer at room temperature for 3 h. The ligation reaction mix (5 μl) was used to transform 50 μl of TOP10 Escherichia coli competent cells (Invitrogen). .. The derived protein sequence of CcapOBP83a-2 was compared and aligned with other members of insect OBPs using software SIAS ( http://imed.med.ucm.es/Tools/sias.html ) with default settings for identity and similarity calculations.

    Ligation:

    Article Title: Production of a recombinant membrane protein in an Escherichia coli strain for the whole cell biosynthesis of phenylacetic acids
    Article Snippet: Afterwards, 6 ng of the styC -containing insert and 16 ng digested pET16bP (molar ratio 4:1) were ligated in 1 × T4 ligase buffer containing 1 U of T4 ligase (Thermo Scientific). .. After a 30 min incubation at 22 °C, ligation product was immediately used for the transformation in strain DH5α as described above.

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins
    Article Snippet: .. Adaptor ligation The treated samples (±Dpn I) were added to 2 µl 10× T4 ligase buffer, 1 µl of 50 µM dsOligos AdRt/AdRb, 2.5 units of T4 DNA ligase (Thermo Fisher Scientific, EL0011) and 6.5 µl H2 O, and incubated overnight at 16-18°C. .. The reaction was cleaned up using an InnuPREP Double EPure Kit (Analytik Jena) and eluted in 50 µl of H2 O.

    Article Title: Reciprocal chromosome translocation associated with TDNA-insertion mutation in Arabidopsis: genetic and cytological analyses of consequences for gametophyte development and for construction of doubly mutant lines
    Article Snippet: Briefly, genomic AtREV3 2/ 2 DNA (200 ng), 0.28 μM “Eco” Adapter DNA, and 0.28 μM “Hind” adapter DNA were digested with 50 U Eco RI endonuclease (New England Biolabs, Ipswich, MA, USA) and 5 U Hin dIII endonuclease (Invitrogen, Carlsbad, CA, USA) in the presence of 5 U T4 DNA-ligase (Invitrogen) for 6 h at 37°C in 1× T4 ligase buffer [40 mM Tris–HCl (pH 7.8 at 25°C), 10 mM MgCl2 , 10 mM dithiothreitol, 0.5 mM ATP (Fermentas, Glen Burnie, MD, USA)]. .. Adapter ligation was completed in a thermocycler: 37°C for 10 s, 10°C for 10 s, repeat for 198 cycles and finished with 20 min at 65°C.

    Article Title: 3C-digital PCR for quantification of chromatin interactions
    Article Snippet: .. To stop the restriction digestion, 1.6% SDS (final concentration) was added, and samples were incubated at 65 °C for 20 min. Ligation were performed at 16 °C for 4 h in 15 mL tubes containing 745 μL 10× T4 ligase Buffer, 10% Triton-X 100, 80 μL 10 mg/mL BSA, 6 mL water, 575 μL of cell lysate, 10 μL 1U/μL T4 ligase (Invitrogen, Grand Island, NY, USA). .. The crosslinks were reversed with Proteinase K (Invitrogen) at 65 °C overnight.

    Article Title: Prostate cancer risk locus at 8q24 as a regulatory hub by physical interactions with multiple genomic loci across the genome
    Article Snippet: .. The restriction endonuclease was inactivated by the addition of SDS to a final concentration of 1.6% and incubated at 65°C for 20 min. Ligation mixes, prepared in 15-mL tubes containing 745 μl of 10× T4 Ligase buffer, 10% Triton-X 100, 80 μl of 10 mg/mL BSA, 6 ml of water, 575 μl of cell lysate and 10 μl of 1 U/μl T4 ligase (Invitrogen, Grand Island, NY), were incubated at 16°C for 4 h and then at room temperature for 30 min. ..

    Article Title: Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata
    Article Snippet: .. The purified PCR product was ligated into pGEM® -T Easy vector (Promega) using a 1:4 M ratio (vector: PCR product) by incubating the mixture with T4-DNA ligase and T4 ligase buffer at room temperature for 3 h. The ligation reaction mix (5 μl) was used to transform 50 μl of TOP10 Escherichia coli competent cells (Invitrogen). .. Positive colonies were selected by their ampicillin resistance, white/blue screening and PCR with gene specific primers.

    Protease Inhibitor:

    Article Title: 3C-digital PCR for quantification of chromatin interactions
    Article Snippet: Each aliquot of cells was lysed with 500 μL 1× cold lysis buffer (10 Mm Tris–HCl Ph 8.0, 10 Mm NaCl, 0.2% Ige cal CA630) including 1× protease inhibitor (Roche, Indianapolis, IN, USA) for at least 15 min on ice. .. To stop the restriction digestion, 1.6% SDS (final concentration) was added, and samples were incubated at 65 °C for 20 min. Ligation were performed at 16 °C for 4 h in 15 mL tubes containing 745 μL 10× T4 ligase Buffer, 10% Triton-X 100, 80 μL 10 mg/mL BSA, 6 mL water, 575 μL of cell lysate, 10 μL 1U/μL T4 ligase (Invitrogen, Grand Island, NY, USA).

    Article Title: Prostate cancer risk locus at 8q24 as a regulatory hub by physical interactions with multiple genomic loci across the genome
    Article Snippet: Two aliquots of cells were lysed with 500 µl of 1× cold lysis buffer (10 m m Tris–HCl, pH 8.0, 10 m m NaCl and 0.2% IGEPAL CA-630) including 1× protease inhibitor (Roche, Indianapolis, IN). .. The restriction endonuclease was inactivated by the addition of SDS to a final concentration of 1.6% and incubated at 65°C for 20 min. Ligation mixes, prepared in 15-mL tubes containing 745 μl of 10× T4 Ligase buffer, 10% Triton-X 100, 80 μl of 10 mg/mL BSA, 6 ml of water, 575 μl of cell lysate and 10 μl of 1 U/μl T4 ligase (Invitrogen, Grand Island, NY), were incubated at 16°C for 4 h and then at room temperature for 30 min.

    Polymerase Chain Reaction:

    Article Title: Reciprocal chromosome translocation associated with TDNA-insertion mutation in Arabidopsis: genetic and cytological analyses of consequences for gametophyte development and for construction of doubly mutant lines
    Article Snippet: T-DNA/genomic junctions were mapped using adapter-ligation-mediated PCR (“anchored-PCR”) as described by , with modifications. .. Briefly, genomic AtREV3 2/ 2 DNA (200 ng), 0.28 μM “Eco” Adapter DNA, and 0.28 μM “Hind” adapter DNA were digested with 50 U Eco RI endonuclease (New England Biolabs, Ipswich, MA, USA) and 5 U Hin dIII endonuclease (Invitrogen, Carlsbad, CA, USA) in the presence of 5 U T4 DNA-ligase (Invitrogen) for 6 h at 37°C in 1× T4 ligase buffer [40 mM Tris–HCl (pH 7.8 at 25°C), 10 mM MgCl2 , 10 mM dithiothreitol, 0.5 mM ATP (Fermentas, Glen Burnie, MD, USA)].

    Article Title: Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata
    Article Snippet: .. The purified PCR product was ligated into pGEM® -T Easy vector (Promega) using a 1:4 M ratio (vector: PCR product) by incubating the mixture with T4-DNA ligase and T4 ligase buffer at room temperature for 3 h. The ligation reaction mix (5 μl) was used to transform 50 μl of TOP10 Escherichia coli competent cells (Invitrogen). .. Positive colonies were selected by their ampicillin resistance, white/blue screening and PCR with gene specific primers.

    DNA Extraction:

    Article Title: Production of a recombinant membrane protein in an Escherichia coli strain for the whole cell biosynthesis of phenylacetic acids
    Article Snippet: Subsequently, the products were purified by gel electrophoresis (1% agarose, 90 V), stained with SYBR Gold (Invitrogen), and isolated from the gel by the DNA Isolation Spin Kit (AppliChem). .. Afterwards, 6 ng of the styC -containing insert and 16 ng digested pET16bP (molar ratio 4:1) were ligated in 1 × T4 ligase buffer containing 1 U of T4 ligase (Thermo Scientific).

    Nucleic Acid Electrophoresis:

    Article Title: Production of a recombinant membrane protein in an Escherichia coli strain for the whole cell biosynthesis of phenylacetic acids
    Article Snippet: Subsequently, the products were purified by gel electrophoresis (1% agarose, 90 V), stained with SYBR Gold (Invitrogen), and isolated from the gel by the DNA Isolation Spin Kit (AppliChem). .. Afterwards, 6 ng of the styC -containing insert and 16 ng digested pET16bP (molar ratio 4:1) were ligated in 1 × T4 ligase buffer containing 1 U of T4 ligase (Thermo Scientific).

    Mutagenesis:

    Article Title: Genetic Manipulation of Campylobacter jejuni
    Article Snippet: .. 5× salt buffer (see recipe)10 mg/ml bovine serum albumin (BSA) 100 mM dithiothreitol (DTT) Donor DNA (containing transposon) Recipient DNA (containing target of mutagenesis or total C. jejuni genomic DNA prepared as in Basic Protocol 6) Transposase: (available from Dr. David Lampe ( ) TE buffer, pH 8.0 ( APPENDIX 2A ) dNTP mix: 1.25 mM each dNTP ( APPENDIX 2A ) T4 DNA polymerase and T4 DNA polymerase buffer (Invitrogen) T4 ligase and T4 ligase buffer (Invitrogen) 30° and 70°C water baths and 11° and 16°C recirculating water baths (or thermal cycler) Nitrocellulose membrane (0.025 μm pore size VSWP (Millipore, cat. no. VSWP04700) Additional reagents and equipment for phenol:chloroform extraction and ethanol precipitation of DNA ( ) .. 1 Prepare transposition reaction by combining the following: 16.0 μl 5× salt buffer 2.0 μl 10 mg/ml BSA 1.6 μl 100 mM DTT 1.0 μg donor DNA 2.0 μg recipient DNA 500 ng transposase Distilled deionized H2 O to 80 μl Incubate reactions at 30°C for 4 hr.

    Isolation:

    Article Title: Production of a recombinant membrane protein in an Escherichia coli strain for the whole cell biosynthesis of phenylacetic acids
    Article Snippet: Subsequently, the products were purified by gel electrophoresis (1% agarose, 90 V), stained with SYBR Gold (Invitrogen), and isolated from the gel by the DNA Isolation Spin Kit (AppliChem). .. Afterwards, 6 ng of the styC -containing insert and 16 ng digested pET16bP (molar ratio 4:1) were ligated in 1 × T4 ligase buffer containing 1 U of T4 ligase (Thermo Scientific).

    Purification:

    Article Title: Production of a recombinant membrane protein in an Escherichia coli strain for the whole cell biosynthesis of phenylacetic acids
    Article Snippet: Subsequently, the products were purified by gel electrophoresis (1% agarose, 90 V), stained with SYBR Gold (Invitrogen), and isolated from the gel by the DNA Isolation Spin Kit (AppliChem). .. Afterwards, 6 ng of the styC -containing insert and 16 ng digested pET16bP (molar ratio 4:1) were ligated in 1 × T4 ligase buffer containing 1 U of T4 ligase (Thermo Scientific).

    Article Title: 3C-digital PCR for quantification of chromatin interactions
    Article Snippet: To stop the restriction digestion, 1.6% SDS (final concentration) was added, and samples were incubated at 65 °C for 20 min. Ligation were performed at 16 °C for 4 h in 15 mL tubes containing 745 μL 10× T4 ligase Buffer, 10% Triton-X 100, 80 μL 10 mg/mL BSA, 6 mL water, 575 μL of cell lysate, 10 μL 1U/μL T4 ligase (Invitrogen, Grand Island, NY, USA). .. 3C samples were then purified using phenol–chloroform extraction and quantified by Qubit dsDNA HS Assay (Life Technologies).

    Article Title: Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata
    Article Snippet: .. The purified PCR product was ligated into pGEM® -T Easy vector (Promega) using a 1:4 M ratio (vector: PCR product) by incubating the mixture with T4-DNA ligase and T4 ligase buffer at room temperature for 3 h. The ligation reaction mix (5 μl) was used to transform 50 μl of TOP10 Escherichia coli competent cells (Invitrogen). .. Positive colonies were selected by their ampicillin resistance, white/blue screening and PCR with gene specific primers.

    Sequencing:

    Article Title: Reciprocal chromosome translocation associated with TDNA-insertion mutation in Arabidopsis: genetic and cytological analyses of consequences for gametophyte development and for construction of doubly mutant lines
    Article Snippet: Briefly, genomic AtREV3 2/ 2 DNA (200 ng), 0.28 μM “Eco” Adapter DNA, and 0.28 μM “Hind” adapter DNA were digested with 50 U Eco RI endonuclease (New England Biolabs, Ipswich, MA, USA) and 5 U Hin dIII endonuclease (Invitrogen, Carlsbad, CA, USA) in the presence of 5 U T4 DNA-ligase (Invitrogen) for 6 h at 37°C in 1× T4 ligase buffer [40 mM Tris–HCl (pH 7.8 at 25°C), 10 mM MgCl2 , 10 mM dithiothreitol, 0.5 mM ATP (Fermentas, Glen Burnie, MD, USA)]. .. PCR amplification was carried out for 10 cycles at 96°C for 20 s and 72°C for 140 s, followed by 15 cycles at 96°C for 20 s and 67°C for 140 s. PCR reactions were diluted 1:5 in distilled H2 O; 1.5 μl of the diluted product was used as the template for a second (“nested”) PCR reaction using primers LBb1 and AP2 ( ): 5 cycles at 96°C for 30 s, 94°C for 20 s and 72°C for 140 s, followed by 23 cycles at 96°C for 20 s, 67°C for 20 s and 72°C for 130 s. Products from the nested PCR reaction were cloned in vector pCR®4 using a TOPO TA Cloning Kit for Sequencing (Invitrogen) and transformed into chemically competent E. coli DH5α by standard procedures.

    Article Title: Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata
    Article Snippet: Paragraph title: Cloning and sequencing ... The purified PCR product was ligated into pGEM® -T Easy vector (Promega) using a 1:4 M ratio (vector: PCR product) by incubating the mixture with T4-DNA ligase and T4 ligase buffer at room temperature for 3 h. The ligation reaction mix (5 μl) was used to transform 50 μl of TOP10 Escherichia coli competent cells (Invitrogen).

    Lysis:

    Article Title: 3C-digital PCR for quantification of chromatin interactions
    Article Snippet: Each aliquot of cells was lysed with 500 μL 1× cold lysis buffer (10 Mm Tris–HCl Ph 8.0, 10 Mm NaCl, 0.2% Ige cal CA630) including 1× protease inhibitor (Roche, Indianapolis, IN, USA) for at least 15 min on ice. .. To stop the restriction digestion, 1.6% SDS (final concentration) was added, and samples were incubated at 65 °C for 20 min. Ligation were performed at 16 °C for 4 h in 15 mL tubes containing 745 μL 10× T4 ligase Buffer, 10% Triton-X 100, 80 μL 10 mg/mL BSA, 6 mL water, 575 μL of cell lysate, 10 μL 1U/μL T4 ligase (Invitrogen, Grand Island, NY, USA).

    Article Title: Prostate cancer risk locus at 8q24 as a regulatory hub by physical interactions with multiple genomic loci across the genome
    Article Snippet: Two aliquots of cells were lysed with 500 µl of 1× cold lysis buffer (10 m m Tris–HCl, pH 8.0, 10 m m NaCl and 0.2% IGEPAL CA-630) including 1× protease inhibitor (Roche, Indianapolis, IN). .. The restriction endonuclease was inactivated by the addition of SDS to a final concentration of 1.6% and incubated at 65°C for 20 min. Ligation mixes, prepared in 15-mL tubes containing 745 μl of 10× T4 Ligase buffer, 10% Triton-X 100, 80 μl of 10 mg/mL BSA, 6 ml of water, 575 μl of cell lysate and 10 μl of 1 U/μl T4 ligase (Invitrogen, Grand Island, NY), were incubated at 16°C for 4 h and then at room temperature for 30 min.

    Nested PCR:

    Article Title: Reciprocal chromosome translocation associated with TDNA-insertion mutation in Arabidopsis: genetic and cytological analyses of consequences for gametophyte development and for construction of doubly mutant lines
    Article Snippet: Briefly, genomic AtREV3 2/ 2 DNA (200 ng), 0.28 μM “Eco” Adapter DNA, and 0.28 μM “Hind” adapter DNA were digested with 50 U Eco RI endonuclease (New England Biolabs, Ipswich, MA, USA) and 5 U Hin dIII endonuclease (Invitrogen, Carlsbad, CA, USA) in the presence of 5 U T4 DNA-ligase (Invitrogen) for 6 h at 37°C in 1× T4 ligase buffer [40 mM Tris–HCl (pH 7.8 at 25°C), 10 mM MgCl2 , 10 mM dithiothreitol, 0.5 mM ATP (Fermentas, Glen Burnie, MD, USA)]. .. PCR amplification was carried out for 10 cycles at 96°C for 20 s and 72°C for 140 s, followed by 15 cycles at 96°C for 20 s and 67°C for 140 s. PCR reactions were diluted 1:5 in distilled H2 O; 1.5 μl of the diluted product was used as the template for a second (“nested”) PCR reaction using primers LBb1 and AP2 ( ): 5 cycles at 96°C for 30 s, 94°C for 20 s and 72°C for 140 s, followed by 23 cycles at 96°C for 20 s, 67°C for 20 s and 72°C for 130 s. Products from the nested PCR reaction were cloned in vector pCR®4 using a TOPO TA Cloning Kit for Sequencing (Invitrogen) and transformed into chemically competent E. coli DH5α by standard procedures.

    Plasmid Preparation:

    Article Title: Production of a recombinant membrane protein in an Escherichia coli strain for the whole cell biosynthesis of phenylacetic acids
    Article Snippet: Afterwards, plasmid DNA was purified by the Gene JET Plasmid Miniprep Kit (Thermo Scientific). .. Afterwards, 6 ng of the styC -containing insert and 16 ng digested pET16bP (molar ratio 4:1) were ligated in 1 × T4 ligase buffer containing 1 U of T4 ligase (Thermo Scientific).

    Article Title: Reciprocal chromosome translocation associated with TDNA-insertion mutation in Arabidopsis: genetic and cytological analyses of consequences for gametophyte development and for construction of doubly mutant lines
    Article Snippet: Briefly, genomic AtREV3 2/ 2 DNA (200 ng), 0.28 μM “Eco” Adapter DNA, and 0.28 μM “Hind” adapter DNA were digested with 50 U Eco RI endonuclease (New England Biolabs, Ipswich, MA, USA) and 5 U Hin dIII endonuclease (Invitrogen, Carlsbad, CA, USA) in the presence of 5 U T4 DNA-ligase (Invitrogen) for 6 h at 37°C in 1× T4 ligase buffer [40 mM Tris–HCl (pH 7.8 at 25°C), 10 mM MgCl2 , 10 mM dithiothreitol, 0.5 mM ATP (Fermentas, Glen Burnie, MD, USA)]. .. PCR amplification was carried out for 10 cycles at 96°C for 20 s and 72°C for 140 s, followed by 15 cycles at 96°C for 20 s and 67°C for 140 s. PCR reactions were diluted 1:5 in distilled H2 O; 1.5 μl of the diluted product was used as the template for a second (“nested”) PCR reaction using primers LBb1 and AP2 ( ): 5 cycles at 96°C for 30 s, 94°C for 20 s and 72°C for 140 s, followed by 23 cycles at 96°C for 20 s, 67°C for 20 s and 72°C for 130 s. Products from the nested PCR reaction were cloned in vector pCR®4 using a TOPO TA Cloning Kit for Sequencing (Invitrogen) and transformed into chemically competent E. coli DH5α by standard procedures.

    Article Title: Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata
    Article Snippet: .. The purified PCR product was ligated into pGEM® -T Easy vector (Promega) using a 1:4 M ratio (vector: PCR product) by incubating the mixture with T4-DNA ligase and T4 ligase buffer at room temperature for 3 h. The ligation reaction mix (5 μl) was used to transform 50 μl of TOP10 Escherichia coli competent cells (Invitrogen). .. Positive colonies were selected by their ampicillin resistance, white/blue screening and PCR with gene specific primers.

    Software:

    Article Title: Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata
    Article Snippet: The purified PCR product was ligated into pGEM® -T Easy vector (Promega) using a 1:4 M ratio (vector: PCR product) by incubating the mixture with T4-DNA ligase and T4 ligase buffer at room temperature for 3 h. The ligation reaction mix (5 μl) was used to transform 50 μl of TOP10 Escherichia coli competent cells (Invitrogen). .. The derived protein sequence of CcapOBP83a-2 was compared and aligned with other members of insect OBPs using software SIAS ( http://imed.med.ucm.es/Tools/sias.html ) with default settings for identity and similarity calculations.

    Agarose Gel Electrophoresis:

    Article Title: Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata
    Article Snippet: The PCR product with the correct size was separated on 1% agarose gel and then excised and purified with the Wizard® SV Gel and PCR Clean-Up System (Promega) following the manufacturer's instructions. .. The purified PCR product was ligated into pGEM® -T Easy vector (Promega) using a 1:4 M ratio (vector: PCR product) by incubating the mixture with T4-DNA ligase and T4 ligase buffer at room temperature for 3 h. The ligation reaction mix (5 μl) was used to transform 50 μl of TOP10 Escherichia coli competent cells (Invitrogen).

    Ethanol Precipitation:

    Article Title: Prostate cancer risk locus at 8q24 as a regulatory hub by physical interactions with multiple genomic loci across the genome
    Article Snippet: The restriction endonuclease was inactivated by the addition of SDS to a final concentration of 1.6% and incubated at 65°C for 20 min. Ligation mixes, prepared in 15-mL tubes containing 745 μl of 10× T4 Ligase buffer, 10% Triton-X 100, 80 μl of 10 mg/mL BSA, 6 ml of water, 575 μl of cell lysate and 10 μl of 1 U/μl T4 ligase (Invitrogen, Grand Island, NY), were incubated at 16°C for 4 h and then at room temperature for 30 min. .. DNA was extracted with phenol–chloroform followed by an ethanol precipitation and quantified by using Qubit dsDNA BR Assay Kits (Life Technologies, Cat# ).

    Article Title: Genetic Manipulation of Campylobacter jejuni
    Article Snippet: .. 5× salt buffer (see recipe)10 mg/ml bovine serum albumin (BSA) 100 mM dithiothreitol (DTT) Donor DNA (containing transposon) Recipient DNA (containing target of mutagenesis or total C. jejuni genomic DNA prepared as in Basic Protocol 6) Transposase: (available from Dr. David Lampe ( ) TE buffer, pH 8.0 ( APPENDIX 2A ) dNTP mix: 1.25 mM each dNTP ( APPENDIX 2A ) T4 DNA polymerase and T4 DNA polymerase buffer (Invitrogen) T4 ligase and T4 ligase buffer (Invitrogen) 30° and 70°C water baths and 11° and 16°C recirculating water baths (or thermal cycler) Nitrocellulose membrane (0.025 μm pore size VSWP (Millipore, cat. no. VSWP04700) Additional reagents and equipment for phenol:chloroform extraction and ethanol precipitation of DNA ( ) .. 1 Prepare transposition reaction by combining the following: 16.0 μl 5× salt buffer 2.0 μl 10 mg/ml BSA 1.6 μl 100 mM DTT 1.0 μg donor DNA 2.0 μg recipient DNA 500 ng transposase Distilled deionized H2 O to 80 μl Incubate reactions at 30°C for 4 hr.

    Concentration Assay:

    Article Title: 3C-digital PCR for quantification of chromatin interactions
    Article Snippet: .. To stop the restriction digestion, 1.6% SDS (final concentration) was added, and samples were incubated at 65 °C for 20 min. Ligation were performed at 16 °C for 4 h in 15 mL tubes containing 745 μL 10× T4 ligase Buffer, 10% Triton-X 100, 80 μL 10 mg/mL BSA, 6 mL water, 575 μL of cell lysate, 10 μL 1U/μL T4 ligase (Invitrogen, Grand Island, NY, USA). .. The crosslinks were reversed with Proteinase K (Invitrogen) at 65 °C overnight.

    Article Title: Prostate cancer risk locus at 8q24 as a regulatory hub by physical interactions with multiple genomic loci across the genome
    Article Snippet: .. The restriction endonuclease was inactivated by the addition of SDS to a final concentration of 1.6% and incubated at 65°C for 20 min. Ligation mixes, prepared in 15-mL tubes containing 745 μl of 10× T4 Ligase buffer, 10% Triton-X 100, 80 μl of 10 mg/mL BSA, 6 ml of water, 575 μl of cell lysate and 10 μl of 1 U/μl T4 ligase (Invitrogen, Grand Island, NY), were incubated at 16°C for 4 h and then at room temperature for 30 min. ..

    DNA Purification:

    Article Title: Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata
    Article Snippet: The purified PCR product was ligated into pGEM® -T Easy vector (Promega) using a 1:4 M ratio (vector: PCR product) by incubating the mixture with T4-DNA ligase and T4 ligase buffer at room temperature for 3 h. The ligation reaction mix (5 μl) was used to transform 50 μl of TOP10 Escherichia coli competent cells (Invitrogen). .. Plasmid DNA containing CcapOBP83a-2 coding region from positive white colonies was extracted using the Wizard® Plus SV Minipreps DNA Purification System (Promega) and then sequenced.

    Staining:

    Article Title: Production of a recombinant membrane protein in an Escherichia coli strain for the whole cell biosynthesis of phenylacetic acids
    Article Snippet: Subsequently, the products were purified by gel electrophoresis (1% agarose, 90 V), stained with SYBR Gold (Invitrogen), and isolated from the gel by the DNA Isolation Spin Kit (AppliChem). .. Afterwards, 6 ng of the styC -containing insert and 16 ng digested pET16bP (molar ratio 4:1) were ligated in 1 × T4 ligase buffer containing 1 U of T4 ligase (Thermo Scientific).

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    Thermo Fisher t4 dna ligase buffer
    T4 Dna Ligase Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher t4 dna ligase
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/Thermo Fisher
    Average 90 stars, based on 1134 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2020-01
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